Anti-ntb-a antibodies and related compositions and methods

ABSTRACT

Disclosed are antibodies, including antibody drug conjugates, that specifically bind to NTB-A. Also disclosed are methods for using the anti-NTB-A antibodies to detect or modulate activity of (e.g., inhibit proliferation of) an NTB-A-expressing cell, as well as for diagnoses or treatment of diseases or disorders (e.g., cancer) associated with NTB-A-expressing cells. Further disclosed is a method of treating multiple myeloma using an anti-NTB-A antibody drug conjugate, which optionally includes an anti-NTB-A antibody as disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.14/758,430 filed Jun. 29, 2015, which is the national stage filing under35 U.S.C. §371 of International Application No. PCT/US2013/077264 filedDec. 20, 2013, which claims the benefit of U.S. Provisional ApplicationNo. 61/745,239 filed Dec. 21, 2012, all of which are incorporated byreference in their entirety for all purposes.

SEQUENCE LISTING

A sequence listing designated NTBA-00112US_ST25.txt of 9 kbytes createdDec. 18, 2013 is incorporated by reference.

BACKGROUND

NTB-A, a single-pass type I membrane glycoprotein also referred to asSLAMF6, is an immunoglobulin superfamily (Ig-SF) member belonging to theCD2/SLAM subfamily. See, e.g., Bottino et al., J. Exp. Med. 194:235-246,2001. NTB-A is characterized, in its extracellular portion, by anN-terminal V-type domain followed by a C2-type domain, while theintracytoplasmic portion contains three tyrosine-based motifs: twoimmunoreceptor tyrosine-based switch motifs (ITSM; TxYxxV/I) and aclassical immunoreceptor tyrosine-based inhibition motif (ITIM;I/V/L/SxYxxL). See id. Through its ITSM motifs, NTB-A associates withthe SH2 domain of the SLAM-associated protein SH2D1A and the relatedEwing's sarcoma activated transcript (EAT) 2. See Bottino et al., supra;Falco et al., Eur. J. Immunol. 34:1663-1672, 2004; Flaig et al., J.Immunol. 172:6524-6527, 2004.

NTB-A is expressed on natural killer (NK) cells, NK-like T-cells,T-cells, monocytes, dendritic cells, B-cells, and eosinophils. SeeSalort JD. et al., Immunology Letters 129-136, 2011; Matesanz-Isabel etal., Immunology Letters 104-112, 2011; Munitz et al., Journal ofImmunology 174:110-118, 2005; Bottino et al., Journal of ExperimentalMedicine 194(3):235-246; 2001. NTB-A can function through homotypicinteractions (i.e., as a self-ligand), and has been shown to act as apositive regulator of NK cell functions via signaling, inducing NK cellcytotoxicity. See, e.g., See Bottino et al., supra; Falco et al., supra;Flaig et al., supra. NTB-A has also been shown to be expressed onB-cells from chronic lymphocytic leukemia (CLL) and B-cell lymphomapatients. See Korver et al., British Journal of Haematology 137:307-318,2007.

SUMMARY OF THE CLAIMED INVENTION

In one aspect, the present invention provides an isolated antibody thatcompetes for specific binding to human NTB-A with a monoclonal antibody(mAb) comprising VH and VL domains having amino acid sequences asrespectively shown in residues 20-135 of SEQ ID NO:1 and residues 21-140of SEQ ID NO:2.

In another aspect, the present invention provides an isolated murineantibody that specifically binds to human NTB-A and comprises VH and VLdomains having amino acid sequences as respectively shown in residues20-135 of SEQ ID NO:1 and residues 21-140 of SEQ ID NO:2, or a chimericor humanized form thereof.

In another aspect, the present invention provides an isolated antibodythat binds to the same epitope on human NTB-A as a mAb comprising VH andVL domains having amino acid sequences as respectively shown in (i)residues 20-135 of SEQ ID NO:1 and residues 21-140 of SEQ ID NO:2, or(ii) residues 20-137 of SEQ ID NO:3 and residues 21-128 of SEQ ID NO:4.

In yet another aspect, the present invention provides an isolatedantibody that specifically binds to human NTB-A and includes (a) a VHdomain comprising an amino acid sequence having at least 80% sequenceidentity with residues 20-135 of SEQ ID NO:1 and a VL domain comprisingan amino acid sequence having at least 80% sequence identity withresidues 21-140 of SEQ ID NO:2, or (b) a VH domain comprising an aminoacid sequence having at least 80% sequence identity with residues 20-137of SEQ ID NO:3 and a VL domain comprising an amino acid sequence havingat least 80% sequence identity with residues 21-128 of SEQ ID NO:4. Insome aspects, such antibody comprises 80% identity to the referencesequences and comprises the same CDRs as the reference sequence.

In still another aspect, the present invention provides an isolatedantibody that specifically binds to human NTB-A and includes VH and VLdomains respectively derived from (a) a VH domain having the amino acidsequence as shown in residues 20-135 of SEQ ID NO:1 and a VL domainhaving the amino acid sequence as shown in residues 21-140 of SEQ IDNO:2, or (b) a VH domain having the amino acid sequence as shown inresidues 20-137 of SEQ ID NO:3 and a VL domain having the amino acidsequence as shown in residues 21-128 of SEQ ID NO:4.

In some embodiments, an antibody of the present invention specificallybinds to human NTB-A and comprises the same CDRs as the VH/VL domainshaving amino acid sequences as respectively shown in residues 20-135 ofSEQ ID NO:1 and residues 21-140 of SEQ ID NO:2. For example, in certainembodiments, the antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2, and CDR-L3 amino acid sequences as respectively shown in SEQ IDNOs:5-10. In other embodiments, the antibody comprises the same CDRs asthe VH/VL domains having amino acid sequences as respectively shown inresidues 20-137 of SEQ ID NO:3 and residues 21-128 of SEQ ID NO:4. Insome such variations, the antibody comprises CDR-H1, CDR-H2, CDR-H3,CDR-LI, CDR-L2, and CDR-L3 amino acid sequences as respectively shown inSEQ ID NOs:11-16.

In other embodiments, an antibody of the present invention specificallybinds to human NTB-A and comprises a set of CDRs (CDRs CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3) having three or fewer amino acidsubstitutions (preferably conservative substitutions) relative to asecond set of CDRs, where the second set of CDRs is from the VH/VLdomains having the amino acid sequences as respectively shown in (i)residues 20-135 of SEQ ID NO:1 and residues 21-140 of SEQ ID NO:2, or(ii) residues 20-137 of SEQ ID NO:3 and residues 21-128 of SEQ ID NO:4.In particular variations, the second set of CDRs comprises CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 amino acid sequences asrespectively shown in SEQ ID NOs:5-10 or SEQ ID NOs:11-16. In someaspects, the antibody competes for specific binding to human NTB-A witha monoclonal antibody (mAb) comprising VH and VL domains having aminoacid sequences as respectively shown in residues 20-135 of SEQ ID NO:1and residues 21-140 of SEQ ID NO:2.

In certain variations, an antibody of the present invention specificallybinds to human NTB-A and is a humanized antibody comprising humanized VHand VL domains. For example, the humanized VH/VL domains may be derivedfrom (i) the VH domain having the amino acid sequence as shown inresidues 20-135 of SEQ ID NO:1 and the VL domain having the amino acidsequence as shown in residues 21-140 of SEQ ID NO:2, respectively, or(ii) the VH domain having the amino acid sequence as shown in residues20-137 of SEQ ID NO:3 and the VL domain having the amino acid sequenceas shown in residues 21-128 of SEQ ID NO:4, respectively. In some suchembodiments, the humanized antibody comprises the same CDRs as the VH/VLdomains specified above. In particular variations, a humanized antibodyderived from the VH and VL domains as specified in (i) above comprisesCDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 amino acid sequencesas respectively shown in SEQ ID NOs:5-10; or a humanized antibodyderived from the VH and VL domains as specified in (ii) above comprisesCDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 amino acid sequencesas respectively shown in SEQ ID NOs:11-16. In other embodiments, thehumanized antibody (i) specifically binds to human NTB-A, (ii) comprisesa set of CDRs (CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3)having three or fewer amino acid substitutions (preferably conservativesubstitutions) relative to a second set of CDRs, where the second set ofCDRs is a set of CDRs as specified above (i.e., the second set of CDRscomprises CDR-HI, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 amino acidsequences as respectively shown in SEQ ID NOs:5-10 and/or SEQ IDNOs:11-16), and (iii) competes for specific binding to human NTB-A witha monoclonal antibody (mAb) comprising VH and VL domains having aminoacid sequences as respectively shown in residues 20-135 of SEQ ID NO:1and residues 21-140 of SEQ ID NO:2.

In some embodiments, an antibody as above further includes at least aportion of an immunoglobulin heavy chain constant region. Theimmunoglobulin heavy chain constant region may be a natural humanconstant region or a genetically engineered variant thereof, such as,for example, a mutant form of a natural human constant region havingreduced binding to an Fcγ receptor relative to the natural humanconstant region (e.g., a variant having the substitutions E233P, L234Vand L235A and/or N297D (numbering according to the EU index as set forthin Kabat)). Suitable heavy chain constant regions include those of humanisotypes IgG1, IgG2, IgG3, and IgG4.

In some embodiments, an antibody as above includes a first polypeptidechain comprising the VH domain and a second polypeptide chain comprisingthe VL domain. In some such variations, the first polypeptide chainfurther includes at least a portion of an immunoglobulin heavy chainconstant region fused to the VH domain, and the second polypeptide chainfurther includes at least a portion of an immunoglobulin light chainconstant region fused to the VL domain. The heavy chain constant regionmay be a natural human constant region or a genetically engineeredvariant thereof, such as, for example, a mutant form of a natural humanconstant region having reduced binding to an Fcy receptor relative tothe natural human constant region. Suitable heavy chain constant regionsinclude those of human isotypes IgG1, IgG2, IgG3, and IgG4.

In certain variations, an antibody as above is conjugated to a cytotoxicor cytostatic agent.

In another aspect, the present invention provides an isolated nucleicacid encoding a VH domain and/or VL domain as defined above. The presentinvention further provides an expression vector comprising apolynucleotide as above, as well as a host cell comprising such anexpression vector and which may be used in methods for producing anantibody of the present invention. Such a method for producing anantibody of the invention typically comprises culturing the host cellunder conditions in which the antibody is expressed and isolating theantibody from the host cell.

In yet another aspect, the present invention provides a pharmaceuticalcomposition comprising an antibody as above and a pharmaceuticallycompatible ingredient.

In still another aspect, the present invention provides a method oftreating a patient having a cancer characterized by NTB-A expression.The treatment method generally includes administering to the patient aneffective regime of an antibody as described above. In certain aspects,the antibody is conjugated to a cytotoxic or cytostatic agent. In someembodiments, the cancer is selected from the group consisting ofmultiple myeloma, acute myeloid leukemia (AML), and a B-cell lymphoma(e.g., non-Hodgkin's lymphoma (NHL)).

In another aspect, the present invention provides a method of treating apatient having multiple myeloma. The treatment method generally includesadministering to the patient an effective regime of an antibody thatspecifically binds to human NTB-A, where the antibody is conjugated to acytotoxic or cytostatic agent. In some variations, the anti-NTB-Aantibody is an antibody as described above.

These and other aspects of the invention will become evident onreference to the following detailed description of the invention and theattached drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an internalization assay using anti-NTB-Aantibody 11A1 on the U-266 multiple myeloma cell line.

FIG. 2 shows the results of an antibody competition assay usinganti-NTB-A antibodies 11A1 and 26B7.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

An “antibody-drug conjugate” refers to an antibody conjugated to acytotoxic agent or cytostatic agent. Typically, antibody-drug conjugatesbind to a target antigen (e.g., NTB-A) on a cell surface followed byinternalization of the antibody-drug conjugate into the cell and releaseof the drug.

A “polypeptide” or “polypeptide chain” is a polymer of amino acidresidues joined by peptide bonds, whether produced naturally orsynthetically. Polypeptides of less than about 10 amino acid residuesare commonly referred to as “peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other nonpeptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “antibody” is used herein to denote immunoglobulin proteinsproduced by the body in response to the presence of an antigen and thatbind to the antigen, as well as antigen-binding fragments and engineeredvariants thereof. Hence, the term “antibody” includes, for example,intact monoclonal antibodies (e.g., antibodies produced using hybridomatechnology) and antigen-binding antibody fragments, such as F(ab′)₂ andFab fragments. Genetically engineered intact antibodies and fragments,such as chimeric antibodies, humanized antibodies, single-chain Fvfragments, single-chain antibodies, diabodies, minibodies, linearantibodies, multivalent or multispecific (e.g., bispecific) hybridantibodies, and the like are also included. Thus, the term “antibody” isused expansively to include any protein that comprises anantigen-binding site of an antibody and is capable of specificallybinding to its antigen. The team “antibody” also includes an antibody byitself (“naked antibody”) or an antibody conjugated to a cytostatic orcytotoxic drug.

The term “genetically engineered antibodies” means antibodies whereinthe amino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics such as, e.g., complement fixation, interaction withcells, and other effector functions. Typically, changes in the variableregion will be made in order to improve the antigen-bindingcharacteristics, improve variable region stability, or reduce the riskof immunogenicity.

An “antigen-binding site of an antibody” is that portion of an antibodythat is sufficient to bind to its antigen. The minimum such region istypically a variable domain or a genetically engineered variant thereof.Single-domain binding sites can be generated from camelid antibodies(see Muyldermans and Lauwereys, J. Mol. Recog. 12:131-140, 1999; Nguyenet al., EMBO J. 19:921-930, 2000) or from VH domains of other species toproduce single-domain antibodies (“dAbs”; see Ward et al., Nature341:544-546, 1989; U.S. Pat. No. 6,248,516 to Winter et al.). In certainvariations, an antigen-binding site is a polypeptide region having only2 complementarity determining regions (CDRs) of a naturally ornon-naturally (e.g., mutagenized) occurring heavy chain variable domainor light chain variable domain, or combination thereof (see, e.g., Pessiet al., Nature 362:367-369, 1993; Qiu et al., Nature Biotechnol.25:921-929, 2007). More commonly, an antigen-binding site of an antibodycomprises both a heavy chain variable (VH) domain and a light chainvariable (VL) domain that bind to a common epitope. Within the contextof the present invention, an antibody may include one or more componentsin addition to an antigen-binding site, such as, for example, a secondantigen-binding site of an antibody (which may bind to the same or adifferent epitope or to the same or a different antigen), a peptidelinker, an immunoglobulin constant region, an immunoglobulin hinge, anamphipathic helix (see Pack and Pluckthun, Biochem. 31:1579-1584, 1992),a non-peptide linker, an oligonucleotide (see Chaudri et al., FEBSLetters 450:23-26, 1999), a cytostatic or cytotoxic drug, and the like,and may be a monomeric or multimeric protein. Examples of moleculescomprising an antigen-binding site of an antibody are known in the artand include, for example, Fv, single-chain Fv (scFv), Fab, Fab′,F(ab′)₂, F(ab)c, diabodies, dAbs, minibodies, nanobodies, Fab-scFvfusions, bispecific (scFv)₄-IgG, and bispecific (scFv)₂-Fab. (See, e.g.,Hu et al., Cancer Res. 56:3055-3061, 1996; Atwell et al., MolecularImmunology 33:1301-1312, 1996; Carter and Merchant, Curr. Opin.Biotechnol. 8:449-454, 1997; Zuo et al., Protein Engineering 13:361-367,2000; and Lu et al., J. Immunol. Methods 267:213-226, 2002.)

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingene(s). One form of immunoglobulin constitutes the basic structuralunit of native (i.e., natural) antibodies in vertebrates. This form is atetramer and consists of two identical pairs of immunoglobulin chains,each pair having one light chain and one heavy chain. In each pair, thelight and heavy chain variable regions (VL and VH) are togetherprimarily responsible for binding to an antigen, and the constantregions are primarily responsible for the antibody effector functions.Five classes of immunoglobulin protein (IgG, IgA, IgM, IgD, and IgE)have been identified in higher vertebrates. IgG comprises the majorclass; it normally exists as the second most abundant protein found inplasma. In humans, IgG consists of four subclasses, designated IgG1,IgG2, IgG3, and IgG4. The heavy chain constant regions of the IgG classare identified with the Greek symbol γ. For example, immunoglobulins ofthe IgG1 subclass contain a γ1 heavy chain constant region. Eachimmunoglobulin heavy chain possesses a constant region that consists ofconstant region protein domains (CH1, hinge, CH2, and CH3; IgG3 alsocontains a CH4 domain) that are essentially invariant for a givensubclass in a species. DNA sequences encoding human and non-humanimmunoglobulin chains are known in the art. (See, e.g., Ellison et al.,DNA 1:11-18, 1981; Ellison et al., Nucleic Acids Res. 10:4071-4079,1982; Kenten et al., Proc. Natl. Acad. Sci. USA 79:6661-6665, 1982; Senoet al., Nuc. Acids Res. 11:719-726, 1983; Riechmann et al., Nature332:323-327, 1988; Amster et al., Nuc. Acids Res. 8:2055-2065, 1980;Rusconi and Kohler, Nature 314:330-334, 1985; Boss et al., Nuc. AcidsRes. 12:3791-3806, 1984; Bothwell et al., Nature 298:380-382, 1982; vander Loo et al., Immunogenetics 42:333-341, 1995; Karlin et al., J. Mol.Evol. 22:195-208, 1985; Kindsvogel et al., DNA 1:335-343, 1982; Breineret al., Gene 18:165-174, 1982; Kondo et al., Eur. J. Immunol.23:245-249, 1993; and GenBank Accession No. J00228.) For a review ofimmunoglobulin structure and function see Putnam, The Plasma Proteins,Vol V, Academic Press, Inc., 49-140, 1987; and Padlan, Mol. Immunol.31:169-217, 1994. The term “immunoglobulin” is used herein for itscommon meaning, denoting an intact antibody, its component chains, orfragments of chains, depending on the context.

Full-length immunoglobulin “light chains” (about 25 kDa or 214 aminoacids) are encoded by a variable region gene at the amino-terminus(encoding about 110 amino acids) and a by a kappa or lambda constantregion gene at the carboxyl-terminus. Full-length immunoglobulin “heavychains” (about 50 kDa or 446 amino acids) are encoded by a variableregion gene (encoding about 116 amino acids) and a gamma, mu, alpha,delta, or epsilon constant region gene (encoding about 330 amino acids),the latter defining the antibody's isotype as IgG, IgM, IgA, IgD, orIgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. (See generally Fundamental Immunology (Paul, ed., RavenPress, N.Y., 2nd ed. 1989), Ch. 7).

An immunoglobulin light or heavy chain variable region (also referred toherein as a “light chain variable domain” (“VL domain”) or “heavy chainvariable domain” (“VH domain”), respectively) consists of a “framework”region interrupted by three hypervariable regions, also called“complementarity determining regions” or “CDRs.” The framework regionsserve to align the CDRs for specific binding to an epitope of anantigen. Thus, the term “hypervariable region” or “CDR” refers to theamino acid residues of an antibody that are primarily responsible forantigen binding. From amino-terminus to carboxyl-terminus, both VL andVH domains comprise the following framework (FR) and CDR regions: FR1,CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989. Kabat alsoprovides a widely used numbering convention (Kabat numbering) in whichcorresponding residues between different heavy chains or betweendifferent light chains are assigned the same number. CDRs 1, 2, and 3 ofa VL domain are also referred to herein, respectively, as CDR-L1,CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred toherein, respectively, as CDR-H1, CDR-H2, and CDR-H3.

Unless the context dictates otherwise, the term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

An immunoglobulin VH or VL domain “derived from” a reference variabledomain means a genetically engineered VH or VL domain comprising some orall CDRs entirely or substantially from the reference variable domain.In some variations, the derived variable domain is a humanized VH or VLdomain. An antibody comprising a VH or VL domain “derived from” areference variable domain will typically retain binding characteristicsof an antibody comprising the reference variable domain.

The term “humanized VH domain” or “humanized VL domain” refers to animmunoglobulin VH or VL domain comprising some or all CDRs entirely orsubstantially from a non-human donor immunoglobulin (e.g., a mouse orrat) and variable domain framework sequences entirely or substantiallyfrom human immunoglobulin sequences. The non-human immunoglobulinproviding the CDRs is called the “donor” and the human immunoglobulinproviding the framework is called the “acceptor.” In some instances,humanized antibodies will retain some non-human residues within thehuman variable domain framework regions to enhance proper bindingcharacteristics (e.g., mutations in the frameworks may be required topreserve binding affinity when an antibody is humanized).

A “humanized antibody” is an antibody comprising one or both of ahumanized VH domain and a humanized VL domain. Immunoglobulin constantregion(s) need not be present, but if they are, they are entirely orsubstantially from human immunoglobulin constant regions.

A CDR in a humanized antibody is “substantially from” a correspondingCDR in a nonhuman antibody when at least 60%, at least 85%, at least90%, at least 95% or 100% of corresponding residues (as defined byKabat) are identical between the respective CDRs. In particularvariations of a humanized VH or VL domain in which CDRs aresubstantially from a non-human immunoglobulin, the CDRs of the humanizedVH or VL domain have no more than six (e.g., no more than five, no morethan four, no more than three, no more than two, or nor more than one)amino acid substitutions (preferably conservative substitutions) acrossall three CDRs relative to the corresponding non-human VH or VL CDRs.The variable region framework sequences of an antibody VH or VL domainor, if present, a sequence of an immunoglobulin constant region, are“substantially from” a human VH or VL framework sequence or humanconstant region, respectively, when at least about 80%, at least 85%, atleast 90%, at least 95%, or 100% of corresponding residues defined byKabat are identical. Hence, all parts of a humanized antibody, exceptthe CDRs, are entirely or substantially from corresponding parts ofnatural human immunoglobulin sequences.

Antibodies are typically provided in isolated form. This means that anantibody is typically at least 50% w/w pure of interfering proteins andother contaminants arising from its production or purification but doesnot exclude the possibility that the antibody is combined with an excessof pharmaceutical acceptable carrier(s) or other vehicle intended tofacilitate its use. Sometimes antibodies are at least 60%, 70%, 80%,90%, 95 or 99% w/w pure of interfering proteins and contaminants fromproduction or purification.

Specific binding of an antibody to its target antigen means an affinityof at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific binding isdetectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces.

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined by X-ray crystallography ofthe antibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other (provided that suchmutations do not produce a global alteration in antigen structure). Twoantibodies have overlapping epitopes if some amino acid mutations thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 55% but preferably 75%,90% or 99% as measured in a competitive binding assay. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Antibodies identified by competition assay also include those thatindirectly compete with a reference antibody by causing a conformationalchange in the target protein thereby preventing binding of the referenceantibody to a different epitope than that bound by the test antibody.

The terms “expression unit” and “expression cassette” are usedinterchangeably herein and denote a nucleic acid segment encoding apolypeptide of interest and capable of providing expression of thenucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. In addition to a transcriptional promoter and terminator,an expression unit may further include other nucleic acid segments suchas, e.g., an enhancer or a polyadenylation signal.

The term “expression vector,” as used herein, refers to a nucleic acidmolecule, linear or circular, comprising one or more expression units.In addition to one or more expression units, an expression vector mayalso include additional nucleic acid segments such as, for example, oneor more origins of replication or one or more selectable markers.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

With regard to proteins as described herein, reference to amino acidresidues corresponding to those specified by SEQ ID NO includespost-translational modifications of such residues.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

The term “effective amount,” in the context of treatment of aNTB-A-expressing disorder by administration of an anti-NTB-A antibody asdescribed herein, refers to an amount of such antibody that issufficient to inhibit the occurrence or ameliorate one or more symptomsof the NTBA-expressing disorder. An effective amount of an antibody isadministered according to the methods of the present invention in an“effective regime.” The term “effective regime” refers to a combinationof amount of the antibody being administered and dosage frequencyadequate to accomplish prophylactic or therapeutic treatment of thedisorder.

For purposes of classifying amino acids substitutions as conservative ornonconservative, the following amino acid substitutions are consideredconservative substitutions: serine substituted by threonine, alanine, orasparagine; threonine substituted by proline or serine; asparaginesubstituted by aspartic acid, histidine, or serine; aspartic acidsubstituted by glutamic acid or asparagine; glutamic acid substituted byglutamine, lysine, or aspartic acid; glutamine substituted by arginine,lysine, or glutamic acid; histidine substituted by tyrosine orasparagine; arginine substituted by lysine or glutamine; methioninesubstituted by isoleucine, leucine or valine; isoleucine substituted byleucine, valine, or methionine; leucine substituted by valine,isoleucine, or methionine; phenylalanine substituted by tyrosine ortryptophan; tyrosine substituted by tryptophan, histidine, orphenylalanine; proline substituted by threonine; alanine substituted byserine; lysine substituted by glutamic acid, glutamine, or arginine;valine substituted by methionine, isoleucine, or leucine; and tryptophansubstituted by phenylalanine or tyrosine. Conservative substitutions canalso mean substitutions between amino acids in the same class. Classesare as follows: Group I (hydrophobic side chains): met, ala, val, leu,ile; Group II (neutral hydrophilic side chains): cys, ser, thr; GroupIII (acidic side chains): asp, glu; Group IV (basic side chains): asn,gln, his, lys, arg; Group V (residues influencing chain orientation):gly, pro; and Group VI (aromatic side chains): trp, tyr, phe.

Two amino acid sequences have “100% amino acid sequence identity” if theamino acid residues of the two amino acid sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinfoiniatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art. (See, e.g., Peruski and Peruski, The Internet andthe New Biology: Tools for Genomic and Molecular Research (ASM Press,Inc. 1997); Wu et al. (eds.), “Information Superhighway and ComputerDatabases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology 123-151 (CRC Press, Inc. 1997); Bishop (ed.), Guide toHuman Genome Computing (2nd ed., Academic Press, Inc. 1998).) Two aminoacid sequences are considered to have “substantial sequence identity” ifthe two sequences have at least 80%, at least 85%, at least 90%, or atleast 95% sequence identity relative to each other.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire variable domain of a heavyor light chain) is being compared with the same region of a referenceantibody, the percentage sequence identity between the subject andreference antibody regions is the number of positions occupied by thesame amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises antibody may contain the antibody alone or incombination with other ingredients.

Designation of a range of values includes all integers within ordefining the range.

An antibody effector function refers to a function contributed by an Fcregion of an Ig. Such functions can be, for example, antibody-dependentcellular cytotoxicity, antibody-dependent cellular phagocytosis, orcomplement-dependent cytotoxicity. Such function can be effected by, forexample, binding of an Fc region to an Fc receptor on an immune cellwith phagocytic or lytic activity or by binding of an Fc region tocomponents of the complement system. Typically, the effect(s) mediatedby the Fc-binding cells or complement components result in inhibitionand/or depletion of the NTB-A-targeted cell. Fc regions of antibodiescan recruit Fc receptor (FcR)-expressing cells and juxtapose them withantibody-coated target cells. Cells expressing surface FcR for IgGsincluding FcγRIII (CD16), FcγRI1 (CD32) and FcγRIII (CD64) can act aseffector cells for the destruction of IgG-coated cells. Such effectorcells include monocytes, macrophages, natural killer (NK) cells,neutrophils and eosinophils. Engagement of FcγR by IgG activatesantibody-dependent cellular cytotoxicity (ADCC) or antibody-dependentcellular phagocytosis (ADCP). ADCC is mediated by CD16⁺ effector cellsthrough the secretion of membrane pore-forming proteins and proteases,while phagocytosis is mediated by CD32⁺ and CD64⁺ effector cells (seeFundamental Immunology, 4^(th) ed., Paul ed., Lippincott-Raven, N.Y.,1997, Chapters 3, 17 and 30; Uchida et al., J. Exp. Med. 199:1659-69,2004; Akewanlop et al., Cancer Res. 61:4061-65, 2001; Watanabe et al.,Breast Cancer Res. Treat. 53:199-207, 1999). In addition to ADCC andADCP, Fc regions of cell-bound antibodies can also activate thecomplement classical pathway to elicit complement-dependent cytotoxicity(CDC). Clq of the complement system binds to the Fc regions ofantibodies when they are complexed with antigens. Binding of Clq tocell-bound antibodies can initiate a cascade of events involving theproteolytic activation of C4 and C2 to generate the C3 convertase.Cleavage of C3 to C3b by C3 convertase enables the activation ofterminal complement components including C5b, C6, C7, C8 and C9.Collectively, these proteins form membrane-attack complex pores on theantibody-coated cells. These pores disrupt the cell membrane integrity,killing the target cell (see Immunobiology, 6^(th) ed., Janeway et al.,Garland Science, N. Y., 2005, Chapter 2).

The term “antibody-dependent cellular cytotoxicity,” or “ADCC,” is amechanism for inducing cell death that depends on the interaction ofantibody-coated target cells with immune cells possessing lytic activity(also referred to as effector cells). Such effector cells includenatural killer cells, monocytes/macrophages and neutrophils. Theeffector cells attach to an Fc region of Ig bound to target cells viatheir antigen-combining sites. Death of the antibody-coated target celloccurs as a result of effector cell activity.

The term “antibody-dependent cellular phagocytosis,” or “ADCP,” refersto the process by which antibody-coated cells are internalized, eitherin whole or in part, by phagocytic immune cells (e.g., macrophages,neutrophils and dendritic cells) that bind to an Fc region of Ig.

The term “complement-dependent cytotoxicity,” or “CDC,” refers to amechanism for inducing cell death in which an Fc region of atarget-bound antibody activates a series of enzymatic reactionsculminating in the formation of holes in the target cell membrane.Typically, antigen-antibody complexes such as those on antibody-coatedtarget cells bind and activate complement component Clq which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes.

A “cytotoxic effect” refers to the depletion, elimination and/or thekilling of a target cell. A “cytotoxic agent” refers to an agent thathas a cytotoxic effect on a cell. Cytotoxic agents can be conjugated toan antibody or administered in combination with an antibody.

A “cytostatic effect” refers to the inhibition of cell proliferation. A“cytostatic agent” refers to an agent that has a cytostatic effect on acell, thereby inhibiting the growth and/or expansion of a specificsubset of cells. Cytostatic agents can be conjugated to an antibody oradministered in combination with an antibody.

The term “pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “pharmaceuticallycompatible ingredient” refers to a pharmaceutically acceptable diluent,adjuvant, excipient, or vehicle with which an anti-NTB-A antibody isformulated.

The phrase “pharmaceutically acceptable salt,” refers topharmaceutically acceptable organic or inorganic salts of an anti-NTB-Aantibody or conjugate thereof or agent administered with an anti-NTB-Aantibody. Exemplary salts include sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p toluenesulfonate, and pamoate (i.e., 1,1′ methylenebis-(2 hydroxy 3 naphthoate)) salts. A pharmaceutically acceptable saltmay involve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counterion. The counterion may be any organic orinorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counterion.

Unless otherwise apparent from the context, when a value is expressed as“about” X or “approximately” X, the stated value of X will be understoodto be accurate to ±10%.

Glycosylation depends on the host cell used to express the antibody.Because the cell type used for expression of recombinant antibodies aspotential therapeutics is rarely the native cell, significant variationsin the glycosylation pattern of the antibodies can occur betweenrecombinantly expressed antibodies in nonnative cells and antibodies ofthe same primary heavy and light chain sequences expressed in theirnative cells. Mammalian cell lines of rodent origin (such as SP2/0, CHOor BHK) are able to confer a glycosylation that has some similarity to ahuman glycosylation. However, some human components may be missing (suchas the 2,6-linked sialylation) and a number of other components notusually found in humans may be present, such as terminals sialic acidsthat do not usually exist in human cells (NeuGc, for example) orterminal galactose linked to another galactose in a way that is usuallyabsent from human cells (Gal-Gal structures). Recombinant IgGs expressedin CHO cells are generally less galactosylated compared to therecombinant immunoglobulins expressed in mouse myeloma cells.Accordingly, recombinant IgGs produced in CHO cells may contain higherlevels of GO glycans compared with rIgGs produced in mouse myeloma celllines.

The glycosylation structure of antibodies can be analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, gel permeationchromatography, monosaccharide compositional analysis, sequentialenzymatic digestion, and High-Performance Anion-Exchange Chromatographywith Pulsed Amperometric Detection, which uses high pH anion exchangechromatography to separate oligosaccharides based on charge. Methods forreleasing oligosaccharides for analytical purposes include enzymatictreatment (commonly performed using peptide-N-glycosidaseF/endo-.beta.-galactosidase), elimination using harsh alkalineenvironment to release mainly O-linked structures, and chemical methodsusing anhydrous hydrazine to release both N- and O-linkedoligosaccharides.

Thus, the glycosylation pattern of a recombinantly expressed antibodycan be characteristic of the cell type in which expression is performed(e.g., CHO) and distinguishable different by any of the above techniquesfrom other cell types particularly cells of other species, such as mouseand human.

DETAILED DESCRIPTION I. General

The present invention provides antibodies that specifically bind toNTB-A. The antibodies are useful, e.g., for treatment and diagnoses ofvarious NTB-A-expressing cancers, as well as for detecting NTB-A (e.g.,detection of NTB-A expression on cells). Methods for such treatment,diagnoses, and NTB-A detection using antibodies of the invention arealso provided.

The present invention also provides a method of treating multiplemyeloma using an antibody-drug conjugate (ADC) that specifically bindsto NTB-A. It is believed that the present inventors are the first todemonstrate killing of multiple myeloma cells using an anti-NTB-A ADCAnti-NTB-A ADCs for treating multiple myeloma may include, for example,ADCs comprising an anti-NTB-A antibody as described herein. In oneaspect, the method comprises administering to a patient in need thereofan antibody that specifically binds to human NTB-A, wherein the antibodyis conjugated to a cytotoxic agent.

II. Target Molecules

Unless otherwise indicated, NTB-A means a human NTB-A. An exemplaryhuman sequence is assigned UniProtKB/Swiss-Prot accession number Q96DU3.Four splice-variant isoforms are known. The mature extracellular regionis bounded by residues 22-226 of Q96DU3.

Unless otherwise apparent from the context, reference to NTB-A means atleast an extracellular domain of the protein and usually the completeprotein other than a cleavable signal peptide (amino acids 1-21 ofQ96DU3).

III. Antibodies of the Invention

In one aspect, the present invention provides isolated anti-NTBAantibodies that specifically bind to an epitope of the mature NTB-Aextracellular region (e.g., an epitope residing within amino acidresidues 22-226 of UniProtKB/Swiss-Prot accession number Q96DU3). Incertain embodiments, an anti-NTB-A antibody in accordance with thepresent invention is capable of competing for binding to human NTB-Aantigen with a monoclonal antibody having the same VH/VL domains as ananti-NTB-A mAb identified and isolated by the present inventors. Incertain aspects, anti-NTB-A antibody in accordance with the presentinvention is a murine antibody as identified herein or a chimeric orhumanized form thereof.

One method of measuring affinity of an antibody for its target antigenis by determining an antibody's apparent dissociation constant. In someaspects, the antibodies described herein have an apparent disassociationconstant (kd) for NTB-A within a range of 0.1 nM to 10 nM, preferably0.1 nM to 5 nM, more preferably 0.1 nM to 2 nM or 0.1 nM to 1 nM.

A mouse anti-NTB-A monoclonal antibody, designated as mAb 11A1, wasidentified and characterized. The murine 11A1 antibody is an IgG1antibody. MAb 11A1 was found to bind to the full-length extracellularregion of NTB-A (residues 22-226 of UniProtKB/Swiss-Prot accessionnumber Q96DU3) with a kd of 0.13 nM, but not to NTB-A isoform 4,(missing residues 18-128 of accession number Q96DU3). A second antibody,designated as mAb 26B7, was also identified and characterized. MAb 26B7was found to compete with mAb 11A1 for binding to human NTB-A in acompetitive binding assay (see Example 5) and bound to NTB-A with a kdof 0.16 nM. The VH, VL, and VH/VL Kabat CDR amino acid sequences foreach of murine mAbs 11A1 and 26B7 were determined and are depicted belowin Tables 1 and 2. The 11A1 and 26B7 antibodies do not bind tocynomolgus monkey NTB-A.

TABLE 1  Anti-NTB-A mAb Variable Region Amino Acid Sequences SEQ mAb ID(VH/VL) Amino Acid Sequence NO: 11AImkcswiifflmavvtgvnsevhlqqsgaelvkpgasvk 1 (VH)lsctasgfnikdyyvhwvkqrt eqglewigkidpedgeikyapkfqgeat it adt s sntaylqlssltsed tavyycarystyfdywgqgttltvss 11AImesntlllwv111wvpgstgdivltqspaslayslgqk 2 (VL)atisckaskkvtifgsisalhwyqqkpgqppkliyngaklesgvsarfsdsgsqnrsqfgnqlsltltidpveadd aatyyclqnkevpytfgggtkleikr 26B7mgwsyiilflvatatgvhsqvqllqpgaevvkpgtsvk 3 (VH)lsckasgynftiywinwvklrpgqglewigdihpgrgntnlnekfktkatltvdtssstaymqlnslafedsalyy carededwyfdvwgagttvtvss 26B7mkllaellglllfcfsgvrcdiqmnqspsslsaslgdt 4 (VL)ititcrasqgisiwfnwyqqksgnipklliyktsnlhtgvpprfsgsgsgtdftltisslqpediatyyclqsqsy pytfgggtkleikr †Amino acidsequences shown include the N-terminal signal peptide (underlined).Reference herein to VH and VL domains for these mAbs are to the maturepolypeptide (and thus do not include the signal peptide), unless thecontext clearly indicates otherwise. ‡Kabat CDRs are shown in bold.

Accordingly, in certain embodiments, the present invention provides anisolated murine comprising (i) a VH domain having the amino acidsequence as shown in residues 20-135 of SEQ ID NO:1 and (ii) a VL domainhaving the amino acid sequence as shown in residues 21-140 of SEQ IDNO:2, or a chimeric or humanized form thereof. As with the murineantibody, the chimeric and humanized forms thereof bind human NTB-A, butnot cynomolgus monkey NTB-A.

In certain embodiments, the present invention provides an isolatedantibody that competes for specific binding to human NTB-A with amonoclonal antibody (mAb) comprising (i) a VH domain having the aminoacid sequence as shown in residues 20-135 of SEQ ID NO:1 and (ii) a VLdomain having the amino acid sequence as shown in residues 21-140 of SEQID NO:2.

The present invention further provides an isolated antibody thatspecifically binds to the same epitope of human NTB-A as a mAbcomprising (a) a VH domain having the amino acid sequence as shown inresidues 20-135 of SEQ ID NO:1 and a VL domain having the amino acidsequence as shown in residues 21-140 of SEQ ID NO:2 or (b) a VH domainhaving the amino acid sequence as shown in residues 20-137 of SEQ IDNO:3 and a VL domain having the amino acid sequence as shown in residues21-128 of SEQ ID NO:4. In certain variations, the antibody binds to thesame epitope of NTB-A as the aforementioned mAb as determined by anepitope mapping method selected from (i) X-ray co-crystallography, (ii)array-based oligo-peptide scanning (also sometimes referred to asoverlapping peptide scan or pepscan analysis), (iii) site-directedmutagenesis (e.g., alanine scanning mutagenesis), and (iv) H/D exchangemass spectrometry. These epitope mapping methods are well-known in theart and may be readily used in accordance with the present invention.

The present invention also provides an isolated antibody thatspecifically binds to human NTB-A and includes (i) a VH domaincomprising an amino acid sequence having at least 80% sequence identitywith residues 20-135 of SEQ ID NO:1 or 20-137 of SEQ ID NO:3; and/or(ii) a VL domain comprising an amino acid sequence having at least 80%sequence identity with residues 21-140 of SEQ ID NO:2 or 21-128 of SEQID NO:4. Typically, where the antibody includes both a heavy chainvariable domain and a light chain variable domain, the VH and VL domainscorrespond to the same reference antibody from Table 1 (i.e., the VH andVL domains typically have specified sequence identities with residues20-135 of SEQ ID NO:1 and 21-140 of SEQ ID NO:2, respectively, or withresidues 20-137 of SEQ ID NO:3 and 21-128 of SEQ ID NO:4, respectively).

The present invention still further provides an isolated antibody thatspecifically binds to human NTB-A and includes (i) a VH domain derivedfrom a VH domain having the amino acid sequence as shown in residues20-135 of SEQ ID NO:1 or 20-137 of SEQ ID NO:3, and/or (ii) a VL domainderived from a VL domain having the amino acid sequence as shown inresidues 21-140 of SEQ ID NO:2 or 21-128 of SEQ ID NO:4. For example,the VH and/or VL domains may be respectively derived from (a) a VHdomain having the amino acid sequence as shown in residues 20-135 of SEQID NO:1 and/or a VL domain having the amino acid sequence as shown inresidues 21-140 of SEQ ID NO:2, or (b) a VH domain having the amino acidsequence as shown in residues 20-137 of SEQ ID NO:3 and/or a VL domainhaving the amino acid sequence as shown in residues 21-128 of SEQ IDNO:4. The variable domain framework sequences of the derived VH or VLdomain may be entirely or substantially from an immunoglobulin sequencedifferent from that of the reference sequence such as, for example, animmunoglobulin sequence from a different species (e.g., human). Thus, incertain embodiments, the present invention provides a humanized antibodycomprising one or both of a humanized VH domain and a humanized VLdomain derived from one or both of the VH and VL domains specified in(a) or (b) above, as further described herein. Typically, but notalways, humanized antibodies will retain some non-human residues fromthe donor species within the human variable domain framework regions.

The present invention still further provides a monoclonal antibody thatspecifically binds to NTB-A and comprises complementary determiningregion (CDR) sequences as set forth in SEQ ID NO:5 (CDR1), SEQ ID NO:6(CDR2), and SEQ ID NO:7 (CDR3), and light chain CDR sequences as setforth in SEQ ID NO:8 (CDR4), SEQ ID NO:9 (CDR5), and SEQ ID NO:10 (CDR6)and having 0, 1, 2 or 3 conservative amino acid substitutions in eachCDR. In some aspects, the antibody is a humanized monoclonal antibody.In some aspects, there are 0 or 1 conservative amino acid substitutionsin each CDR. Such an antibody can be for example, mouse, chimeric,humanized or veneered.

The present invention still further provides a monoclonal antibody thatspecifically binds to NTB-A and comprises complementary determiningregion (CDR) sequences as set forth in SEQ ID NO:11 (CDR1), SEQ ID NO:12(CDR2), and SEQ ID NO:13 (CDR3), and light chain CDR sequences as setforth in SEQ ID NO:14 (CDR4), SEQ ID NO:15 (CDR5), and SEQ ID NO:16(CDR6) and having 0, 1, 2 or 3 conservative amino acid substitutions ineach CDR. In some aspects, the antibody is a humanized monoclonalantibody. Such an antibody can be for example, mouse, chimeric,humanized or veneered.

The antibodies of the present invention may be assayed for specificbinding by any method known in the art. The immunoassays which can beused include, but are not limited to, assay systems using techniquessuch as western blots, radioimmunoassays, ELISA, “sandwich”immunoassays, immunoprecipitation assays, precipitin assays, geldiffusion precipitin assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, and complement-fixation assays.Such assays are routine and well-known in the art (see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & sons, Inc., New York). Routine assays such as those described inAntibodies, A Laboratory Manual (Cold Spring Harbor Laboratory, EdHarlow and David Lane, 1988) can also be performed. Additionally, theBIACORE® (GE Healthcare, Piscataway, N.J.) is only one of a variety ofsurface plasmon resonance assay formats that are routinely used forbinding analysis of monoclonal antibodies. Other references, e.g., TheEpitope Mapping Protocols, Methods in Molecular Biology, Vol. 66 (GlennMorris ed. Humana Press, 1996), describe alternative methods that couldbe used to bind antibodies and would be expected to provide comparableinformation regarding the binding specificity of the antibodies toNTB-A.

To evaluate whether an antibody competes for specific binding to humanNTB-A with a mAb comprising the VH and VL domains of the 11A1 mAb (i.e.,a VH domain having the amino acid sequence as shown in residues 20-135of SEQ ID NO:1 and a VL domain having the amino acid sequence as shownin residues 21-140 of SEQ ID NO:2), a competitive binding assay asfollows is used.

This assay utilizes a “reference antibody” comprising the 11A1 murineIgG1 antibody (i.e., the 11A1 VH and VL domains in the bivalentstructure of a native antibody). NTB-A positive Ramos cells are platedat 2×10⁵ cells per well in a 96 well V-bottom plate (Thermo Scientific,Rochester, N.Y.). Five-fold serial dilutions of 2× concentratedantibodies (20 μg/ml is the 2× starting concentration unlabeled sampleantibodies) are prepared in FACs buffer (PBS+2% fetal bovine serum+0.02%sodium azide) containing a 2× constant concentration of AF647 labeled11A1 murine antibody at 2× its Kd value of 0.0188 μg/mL (0.0376 μg/mL).The antibody solutions are incubated with cells for 1 hour on ice,protected from light. The cells are washed twice with FACs buffer andanalyzed on the LSRII flow cytometer (BD BioSciences, San Jose, Calif.).Data are presented as percent of maximum binding. The sample antibody“competes with” the labeled reference antibody for specific binding toNTB-A if the sample antibody reduces the reference antibody's binding toNTB-A to levels less than 45% of maximum binding (i.e., 0 to 45%) at aconcentration of 10 μg/ml of unlabeled sample antibody. In some aspects,an antibody competes for binding if the sample antibody reduces thereference antibody's binding to NTB-A to levels less than 30% of maximumbinding at a concentration of 10 μg/ml of unlabeled sample antibody. Insome aspects, an antibody competes for binding if the sample antibodyreduces the reference antibody's binding to NTB-A to levels less than20% of maximum binding at a concentration of 10 μg/ml of unlabeledsample antibody. In some aspects, an antibody competes for binding ifthe sample antibody reduces the reference antibody's binding to NTB-A tolevels less than 10% of maximum binding at a concentration of 10 μg/mlof unlabeled sample antibody. In some aspects, an antibody competes forbinding if the sample antibody reduces the reference antibody's bindingto NTB-A to levels less than 5% of maximum binding at a concentration of10 μg/ml of unlabeled sample antibody. In some aspects, an antibodycompetes for binding if the sample antibody reduces the referenceantibody's binding to NTB-A to levels less than 2% or less than 1% ofmaximum binding at a concentration of 10 μg/ml of unlabeled sampleantibody. As can be seen from FIG. 2, the murine 11A1 antibody competesfor binding with itself and with the 26B7 antibody. It does not competefor binding with the NT-7 antibody.

In some embodiments, an anti-NTBA antibody includes a heavy chainvariable domain and/or light chain variable domain that is substantiallyidentical to the heavy and/or light chain variable domain(s) of anantibody listed in Table 1.

Accordingly, in certain embodiments, an anti-NTB-A antibody has (a) aheavy chain variable domain that is at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or at least 99.5% identical to the amino acid sequence of a VHdomain listed in Table 1 and/or (b) a light chain variable domain thatis at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of a VL domain listed in Table 1. In particularvariations, the anti-NTB-A antibody includes (a) a heavy chain variabledomain having the amino acid sequence of a VH domain listed in Table 1and/or (b) a light chain variable domain having the amino acid sequenceof a VL domain listed in Table 1. In some embodiments where an antibodycomprises both a heavy chain variable domain and a light chain variabledomain, the heavy and light chain variable domains correspond to thesame reference antibody from Table 1. For example, in more specificvariations, an anti-NTB-A antibody comprises light and heavy chainvariable domains having respective VH and VL amino acid sequencesselected from the following VH/VL sequence pairs: (I) residues 20-135 ofSEQ ID NO:1 and residues 21-140 of SEQ ID NO:2; and (II) residues 20-137of SEQ ID NO:3 and residues 21-128 of SEQ ID NO:4.

In some embodiments, an anti-NTB-A antibody of the present inventioncomprises one or more CDRs of an anti-NTB-A antibody listed in Table 1.The Kabat CDRs of the VH and VL domains from Table 1 are also set forthbelow in Table 2.

TABLE 2  CDR Sequences for Anti-NTB-A Antibody VH and VL Domains mAb CDRAmino Acid Sequence SEO ID NO: 11A1 CDR-H1 dyyvh 5 CDR-H2kidpedgeikyapkfqg 6 CDR-H3 ystyfdy 7 CDR-L1 kaskkvtifgsisalh 8 CDR-L2ngakles 9 CDR-L3 lqnkevpyt 10 26B7 CDR-H1 iywin 11 CDR-H2dihpgrgntnlnekfkt 12 CDR-H3 ededwyfdv 13 CDR-L1 rasqgisiwfn 14 CDR-L2ktsnlht 15 CDR-L3 1qsqsypyt 16

For example, in certain variations, the antibody comprises a heavy chainCDR (at least one of the CDR-H1, CDR-H2, and CDR-H3 regions) and/or acorresponding light chain CDR (at least one of the CDR-L1, CDR-L2, andCDR-L3 regions) of an antibody listed in Table 1. In typicalembodiments, the anti-NTB-A antibody has two or three heavy chain CDRsand/or two or three light chain CDRs of an antibody listed in Table 1.In some variations, where an anti-NTB-A antibody has at least one heavychain CDR of an antibody listed in Table 1, the antibody furthercomprises at least one corresponding light chain CDR.

In particular variations, an anti-NTB-A antibody includes a heavy and/orlight chain variable domain, the heavy or light chain variable domainhaving (a) a set of three CDRs corresponding to the heavy or light chainCDRs as shown for an antibody listed in Table 1, and (b) a set of fourframework regions. For example, an anti-NTB-A antibody can include aheavy and/or light chain variable domain, where the heavy or light chainvariable domain has (a) a set of three CDRs, in which the set of CDRsare from an antibody listed in Table 1, and (b) a set of four frameworkregions, in which the set of framework regions are identical to ordifferent (e.g., from a human framework region) from the set offramework regions of the same antibody listed in Table 1.

In certain variations, an anti-NTB-A antibody includes both (I) a heavychain variable domain having (a) a set of three CDRs corresponding tothe heavy chain CDRs as shown for an antibody listed in Table 1, and (b)a set of four framework regions; and (II) a light chain variable domainhaving (a) a set of three CDRs corresponding to the light chain CDRs asshown for an antibody listed in Table 1, and (b) a set of four frameworkregions. In typical embodiments, both the heavy chain and light chainCDRs are from the same antibody listed in Table 1.

In some embodiments, an anti-NTB-A antibody in accordance with thepresent invention includes a heavy and/or light chain variable regioncomprising at least one CDR having zero, one, two, three, or four aminoacid substitutions (preferably conservative substitutions) relative to aCDR of a VL or VH domain listed in Table 1. In certain variations, forexample, an anti-NTB-A antibody in accordance with the present inventioncomprises heavy chain CDRs CDR1-H1, CDR-H2, and CDR-H3, wherein at leastone of CDR-H1, CDR2-H2, and CDR-H3 comprises zero, one, two, three, orfour amino acid substitutions (preferably conservative substitutions)relative to a VH domain in Table 1. In other variations, an anti-NTB-Aantibody in accordance with the present invention comprises light chainCDRs CDR1-L1, CDR2-L2, and CDR3-L3, wherein at least one of CDR1-L1,CDR2-L2, and CDR3-L3 comprises zero, one, two, three, or four amino acidsubstitutions (preferably conservative substitutions) relative to a VLdomain listed in Table 1. In certain embodiments, an anti-NTB-A antibodycomprises both sets of heavy chain and light chain CDRs as above.Particularly suitable anti-NTB-A antibodies comprise a light chainvariable domain comprising CDRs CDR1-L1, CDR2-L2, and CDR3-L3 and aheavy chain variable domain comprising CDRs CDR1-H1, CDR2-H2, andCDR3-H3, wherein said set of heavy and light chain CDRs has six orfewer, typically five or fewer, more typically four or fewer, and mosttypically 3 or fewer amino acid substitutions (preferably conservativesubstitutions) relative to CDRs from a VH domain and a VL domain listedin Table 1; in some such variations, the six or fewer amino acidsubstitutions are relative to CDRs from a VH domain and a VL domain ofthe same antibody listed in Table 1.

In certain embodiments, an anti-NTB-A antibody of the present inventioncomprises a humanized VH domain and/or a humanized VL domainrespectively derived from a VH and/or VL domain listed in Table 1. Inparticular variations, the anti-NTB-A antibody comprises both ahumanized VH domain and a humanized VL domain respectively derived froma VH domain and a VL domain listed in Table 1. Typically, the humanizedVH and VL domains are derived from the same antibody listed in Table 1.

A humanized antibody is a genetically engineered antibody in which CDRsfrom a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. Thus, a humanized antibody is an antibody having some or allCDRs entirely or substantially from a donor antibody and variable regionframework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Similarly, a humanized VHdomain has at least one, two, and usually all three CDRs entirely orsubstantially from a donor antibody VH domain, and variable regionframework sequences entirely or substantially from a human antibody VHdomain; such a humanized VH domain may be linked, typicallyamino-terminal to, an immunoglobulin heavy chain constant regionentirely or substantially from a human heavy chain constant regionsequence. Similarly, a humanized VL domain has at least one, two, andusually all three CDRs entirely or substantially from a donor antibodyVL domain, and variable region framework sequences entirely orsubstantially from a human antibody VL domain; such a humanized VLdomain may be linked, typically amino-terminal to, an immunoglobulinlight chain constant region entirely or substantially from a human lightchain constant region sequence. Typically, a humanized antibodycomprises both a humanized VH domain and a humanized VL domain.Generally, but not always, humanized antibodies will retain somenon-human residues within the human variable domain framework regions toenhance proper binding characteristics (e.g., mutations in theframeworks may be required to preserve binding affinity when an antibodyis humanized).

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a non-human antibody, they can also be madewith less than all CDRs (e.g., at least 3, 4, or 5) CDRs from anon-human antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002;Vajdos et al., J. Mol. Biol., 320: 415-428, 2002; Iwahashi et al., Mol.Immunol. 36:1079-1091, 1999; Tamura et al., J. Immunol., 164:1432-1441,2000). In some aspects, the humanized antibodies will incorporate allsix CDRs but will have zero, one, two, or three conservativesubstitutions in one or more of the CDRs.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region,

(3) otherwise interacts with a CDR region (e.g., is within about 6 Å ofa CDR region); or

(4) mediates interaction between the heavy and light chains.

Another possible variation is to substitute certain residues in the CDRsof the mouse antibody with corresponding residues from human CDRsequences, typically from the CDRs of the human acceptor sequences usedin designing the exemplified humanized antibodies. In some antibodiesonly part of the CDRs, namely the subset of CDR residues required forbinding, termed the SDRs, are needed to retain binding in a humanizedantibody. CDR residues not contacting antigen and not in the SDRs can beidentified based on previous studies (for example, residues H60-H65 inCDR H2 are often not required), from regions of Kabat CDRs lying outsideChothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), bymolecular modeling, and/or empirically, or as described in Gonzales etal., Mol. Immunol. 41: 863, 2004. In such humanized antibodies atpositions in which one or more donor CDR residues is absent or in whichan entire donor CDR is omitted, the amino acid occupying the positioncan be an amino acid occupying the corresponding position (by Kabatnumbering) in the acceptor antibody sequence. The number of suchsubstitutions of acceptor for donor amino acids in the CDRs to includereflects a balance of competing considerations. Such substitutions arepotentially advantageous in decreasing the number of mouse amino acidsin a humanized antibody and consequently decreasing potentialimmunogenicity. However, substitutions can also cause changes ofaffinity, and significant reductions in affinity are preferably avoided.In a further variation, one or more residues in a CDR of a humanizedanti-NTB-A antibody (which would otherwise be the same as the CDR of anantibody listed in Table 1) can be replaced by corresponding residuesfrom a CDR from a different antibody listed in Table 1. Positions forsubstitution within CDRs and amino acids to substitute can also beselected empirically.

Although not preferred, other amino acid substitutions can be made, forexample, in framework residues not in contact with the CDRs, or evensome potential CDR-contact residues or amino acids within the CDRs.Often the replacements made in the variant humanized sequences areconservative with respect to the replaced amino acids. Preferably, suchreplacements, whether or not conservative, have no substantial effect onthe binding affinity or potency of the humanized antibody, that is, itsability to bind human NTB-A or inhibit growth of cancer cells.

Preferred anti-NTB-A antibodies or conjugated forms thereof (e.g.,antibody-drug conjugates) inhibit cancer (e.g., growth of cells,metastasis and/or lethality to the organisms) as shown on cancerouscells propagating in culture, in an animal model, or in a clinicaltrial. Animal models can be formed by implanting NTB-A-expressing humantumor cell lines into appropriate immunodeficient rodent strains, e.g.,athymic nude mice or SCID mice. These tumor cell lines can beestablished in immunodeficient rodent hosts either as solid tumor bysubcutaneous injections or as disseminated tumors by intravenousinjections. Once established within a host, these tumor models can beapplied to evaluate the therapeutic efficacies of the anti-NTB-Aantibodies or conjugated forms thereof.

In certain variations, an anti-NTBA antibody of the present inventioncomprises a VH and/or VL domain linked to at least a portion of animmunoglobulin constant region (e.g., a human immunoglobulin constantregion). For example, in some embodiments, the anti-NTB-A antibodycomprises first and second polypeptide chains, where the firstpolypeptide chain comprises a VH domain as described herein linked to atleast a portion of an immunoglobulin heavy chain constant region and thesecond polypeptide chain comprises a VL domain as described hereinlinked to at least a portion of an immunoglobulin light chain constantregion. Typically, the VH or VL domain is linked amino-terminal to animmunoglobulin constant region or portion thereof. In particularvariations of an antibody comprising first and second polypeptidechains, the first and second polypeptide chains have a domain structurecorresponding to the heavy and light chains of an intact nativeantibody, e.g., a first polypeptide (heavy) chain having theamino-terminal to carboxyl-terminal domain structure ofVH-CH1-hinge-CH2-CH3 and a second polypeptide (light) chain having theamino-terminal to carboxyl-terminal domain structure of VL-CL.

In other embodiments, the anti-NTB-A antibody is a single-chain antibodycomprising a VH domain, a VL domain, and at least a portion of animmunoglobulin constant region (e.g., a heavy chain constant regionlacking a CH1 domain) linked within a single polypeptide chain. Forexample, the VH and VL domains may be constructed as a single-chain Fv(scFv) in either a VH/VL or VL/VH (amino-terminal/carboxyl-terminal)orientation, with the scFv linked (typically amino-terminal) to a heavychain constant region, such as, e.g., a constant region comprising theCH2 and CH3 domains but lacking the CH1 domain. The scFv is typicallylinked to the constant region via a linker such as, for example, alinker derived from an immunoglobulin hinge region.

The choice of constant region can depend, in part, whetherantibody-dependent cell-mediated cytotoxicity, antibody dependentcellular phagocytosis and/or complement dependent cytotoxicity aredesired. For example, human isotopes IgG1 and IgG3 have strongcomplement-dependent cytotoxicity, human isotype IgG2 weakcomplement-dependent cytotoxicity and human IgG4 lackscomplement-dependent cytotoxicity. Human IgG1 and IgG3 also inducestronger cell mediated effector functions than human IgG2 and IgG4.Light chain constant regions can be lambda or kappa. Antibodies can beexpressed, e.g., as tetramers containing two light and two heavy chains,as separate heavy chains, light chains, as Fab, Fab′, F(ab′)2, and Fv,or as single chain antibodies in which heavy and light chain variabledomains are linked through a spacer. Additionally, the constant regionscan be mutated, if desired. In some aspects, a mutant form of a naturalhuman constant region will have reduced binding to an Fcγ receptorrelative to the natural human constant region.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype binds to a non-polymorphic region of a one or more otherisotypes.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., Biol. Chem.279:6213, 2004).

Exemplary substitution include the amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position234, 235, 237, 239, 267, 298, 299, 326, 330, or 332, preferably an S239Cmutation in a human IgG1 isotype (US 20100158909). In some aspects, thepresence of an additional cysteine residue allows interchain disulfidebond formation. Such interchain disulfide bond formation can causesteric hindrance, thereby reducing the affinity of the Fc region-FcγRbinding interaction. The cysteine residue(s) introduced in or inproximity to the Fc region of an IgG constant region can also serve assites for conjugation to therapeutic agents (i.e., coupling cytotoxicdrugs using thiol specific reagents such as maleimide derivatives ofdrugs. The presence of a therapeutic agent causes steric hindrance,thereby further reducing the affinity of the Fc region-FcγR bindinginteraction. Other substitutions at any of positions 234, 235, 236and/or 237 reduce affmity for Fcγ receptors, particularly FcγRI receptor(see, e.g., U.S. Pat. No. 6,624,821, U.S. Pat. No. 5,624,821.)

The in vivo half-life of an antibody can also impact on its effectorfunctions. The half-life of an antibody can be increased or decreased tomodify its therapeutic activities. FcRn is a receptor that isstructurally similar to MHC Class I antigen that non-covalentlyassociates with β2-microglobulin. FcRn regulates the catabolism of IgGsand their transcytosis across tissues (Ghetie and Ward, Annu. Rev.Immunol. 18:739-766, 2000; Ghetie and Ward, Immunol. Res. 25:97-113,2002). The IgG-FcRn interaction takes place at pH 6.0 (pH ofintracellular vesicles) but not at pH 7.4 (pH of blood); thisinteraction enables IgGs to be recycled back to the circulation (Ghetieand Ward, 2000, supra; Ghetie and Ward, 2002, supra). The region onhuman IgG1 involved in FcRn binding has been mapped (Shields et al., J.Biol. Chem. 276:6591-604, 2001). Alanine substitutions at positionsPro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 ofhuman IgG1 enhance FcRn binding (Shields et al., supra). IgG1 moleculesharboring these substitutions have longer serum half-lives.Consequently, these modified IgG1 molecules may be able to carry outtheir effector functions, and hence exert their therapeutic efficacies,over a longer period of time compared to unmodified IgG1. Otherexemplary substitutions for increasing binding to FcRn include a Gln atposition 250 and/or a Leu at position 428. EU numbering is used for allposition in the constant region.

Oligosaccharides covalently attached to the conserved Asn297 areinvolved in the ability of the Fc region of an IgG to bind FcγR (Lund etal., J. Immunol. 157:4963-69, 1996; Wright and Morrison, TrendsBiotechnol. 15:26-31, 1997). Engineering of this glycoform on IgG cansignificantly improve IgG-mediated ADCC. Addition of bisectingN-acetylglucosamine modifications (Umana et al., Nat. Biotechnol.17:176-180, 1999; Davies et al., Biotech. Bioeng. 74:288-94, 2001) tothis glycoform or removal of fucose (Shields et al., J. Biol. Chem.277:26733-40, 2002; Shinkawa et al., J. Biol. Chem. 278:6591-604, 2003;Niwa et al., Cancer Res. 64:2127-33, 2004) from this glycoform are twoexamples of IgG Fc engineering that improves the binding between IgG Fcand FcγR, thereby enhancing Ig-mediated ADCC activity.

A systemic substitution of solvent-exposed amino acids of human IgG1 Fcregion has generated IgG variants with altered FcγR binding affinities(Shields et al., J. Biol. Chem. 276:6591-604, 2001). When compared toparental IgG1, a subset of these variants involving substitutions atThr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 toAla demonstrate increased in both binding affinity toward FcγR and ADCCactivity (Shields et al., 2001, supra; Okazaki et al., J. Mol. Biol.336:1239-49, 2004).

Complement fixation activity of antibodies (both Clq binding and CDCactivity) can be improved by substitutions at Lys326 and Glu333(Idusogie et al., J. Immunol. 166:2571-2575, 2001). The samesubstitutions on a human IgG2 backbone can convert an antibody isotypethat binds poorly to Clq and is severely deficient in complementactivation activity to one that can both bind Clq and mediate CDC(Idusogie et al., supra). Several other methods have also been appliedto improve complement fixation activity of antibodies. For example, thegrafting of an 18-amino acid carboxyl-terminal tail piece of IgM to thecarboxyl-termini of IgG greatly enhances their CDC activity. This isobserved even with IgG4, which normally has no detectable CDC activity(Smith et al., J. Immunol. 154:2226-36, 1995). Also, substituting Ser444located close to the carboxy-terminal of IgG1 heavy chain with Cysinduced tail-to-tail dimerization of IgG1 with a 200-fold increase ofCDC activity over monomeric IgG1 (Shopes et al., J. Immunol.148:2918-22, 1992). In addition, a bispecific diabody construct withspecificity for Clq also confers CDC activity (Kontermann et al., Nat.Biotech. 15:629-31, 1997).

Complement activity can be reduced by mutating at least one of the aminoacid residues 318, 320, and 322 of the heavy chain to a residue having adifferent side chain, such as Ala. Other alkyl-substituted non-ionicresidues, such as Gly, Ile, Leu, or Val, or such aromatic non-polarresidues as Phe, Tyr, Trp and Pro in place of any one of the threeresidues also reduce or abolish Clq binding. Ser, Thr, Cys, and Met canbe used at residues 320 and 322, but not 318, to reduce or abolish Clqbinding activity. Replacement of the 318 (Glu) residue by a polarresidue may modify but not abolish Clq binding activity. Replacingresidue 297 (Asn) with Ala results in removal of lytic activity but onlyslightly reduces (about three fold weaker) affinity for Clq. Thisalteration destroys the glycosylation site and the presence ofcarbohydrate that is required for complement activation. Any othersubstitution at this site also destroys the glycosylation site. Thefollowing mutations and any combination thereof also reduce Clq binding:D270A, K322A, P329A, and P311S (see WO 06/036291).

Reference to a human constant region includes a constant region with anynatural allotype or any permutation of residues occupying polymorphicpositions in natural allotypes. Also, up to 1, 2, 5, or 10 mutations maybe present relative to a natural human constant region, such as thoseindicated above to reduce Fcgamma receptor binding or increase bindingto FcRn.

IV. Nucleic Acids and Methods of Production

The invention further provides nucleic acids encoding any of the VHand/or VL domains described above, including polypeptides comprising theVH and/or VL domains linked to additional polypeptide segments such, forexample, polypeptide segments corresponding to an immunoglobulinconstant region. Typically, the nucleic acids also encode a signalpeptide fused amino-terminal to the mature polypeptide comprising the VHand/or VL domains. Coding sequences on nucleic acids can be in operablelinkage with regulatory sequences to ensure expression of the codingsequences, such as a promoter, enhancer, ribosome binding site,transcription termination signal and the like. The nucleic acids canoccur in isolated form or can be cloned into one or more vectors. Thenucleic acids can be synthesized by for example, solid state synthesisor PCR of overlapping oligonucleotides. Nucleic acids encoding both a VHdomain and a VL domain (e.g., in the context of antibodies comprisingseparate heavy and light chains) can be joined as one contiguous nucleicacid, e.g., within an expression vector, or can be separate, e.g., eachcloned into its own expression vector.

Anti-NTB-A antibodies are typically produced by recombinant expressionof one or more nucleic acids encoding one or more antibody chains.Recombinant polynucleotide constructs typically include an expressioncontrol sequence operably linked to the coding sequences of one or morepolypeptide chains comprising VH and/or VL domains, includingnaturally-associated or heterologous promoter regions. Preferably, theexpression control sequences are eukaryotic promoter systems in vectorscapable of transforming or transfecting eukaryotic host cells. Once thevector has been incorporated into the appropriate host, the host ismaintained under conditions suitable for high level expression of thenucleotide sequences, and the collection and purification of thecross-reacting antibodies.

In certain embodiments for the expression of antibodies comprising firstand second polypeptide chains (e.g., heavy and light chains), the twopolypeptide chains are co-expressed from separate vectors in the hostcell for expression of the entire antibody molecule. In otherembodiments for the expression of double-chained antibodies, the twopolypeptide chains are co-expressed from separate expression units inthe same vector in the host cell for expression of the entire antibodymolecule.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines (e.g., DG44), variousCOS cell lines, HeLa cells, HEK293 cells, L cells, andnon-antibody-producing myelomas including Sp2/0 and NS0. Preferably, thecells are non-human. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, an enhancer (Queen et al., Immunol. Rev. 89:49, 1986), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149, 1992.

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

V. Antibody Drug Conjugates

Anti-NTB-A antibodies can be conjugated to cytotoxic or cytostaticmoieties (including pharmaceutically compatible salts thereof) to forman antibody drug conjugate (ADC). Particularly suitable moieties forconjugation to antibodies are cytotoxic agents (e.g., chemotherapeuticagents), prodrug converting enzymes, radioactive isotopes or compounds,or toxins (these moieties being collectively referred to as atherapeutic agent). For example, an anti-NTB-A antibody can beconjugated to a cytotoxic agent such as a chemotherapeutic agent, or atoxin (e.g., a cytostatic or cytocidal agent such as, for example,abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin). Examples ofuseful classes of cytotoxic agents include, for example, DNA minorgroove binders, DNA alkylating agents, and tubulin inhibitors. Exemplarycytotoxic agents include, for example, auristatins, camptothecins,calicheamicins, duocarmycins, etoposides, maytansinoids (e.g., DM1, DM2,DM3, DM4), taxanes, benzodiazepines (e.g., pyrrolo[1,4]benzodiazepines,indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vincaalkaloids.

An anti-NTB-A antibody can be conjugated to a pro-drug convertingenzyme. The pro-drug converting enzyme can be recombinantly fused to theantibody or chemically conjugated thereto using known methods. Exemplarypro-drug converting enzymes are carboxypeptidase G2, betaglucuronidase,penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase,nitroreductase and carboxypeptidase A.

Techniques for conjugating therapeutic agents to proteins, and inparticular to antibodies, are well-known. (See, e.g., Alley et al.,Current Opinion in Chemical Biology 2010 14:1-9; Senter, Cancer J.,2008, 14(3):154-169.) The therapeutic agent can be conjugated in amanner that reduces its activity unless it is cleaved off the antibody(e.g., by hydrolysis, by proteolytic degradation, or by a cleavingagent). In some aspects, the therapeutic agent is attached to theantibody with a cleavable linker that is sensitive to cleavage in theintracellular environment of the NTB-A-expressing cancer cell but is notsubstantially sensitive to the extracellular environment, such that theconjugate is cleaved from the antibody when it is internalized by theNTB-A-expressing cancer cell (e.g., in the endosomal or, for example byvirtue of pH sensitivity or protease sensitivity, in the lysosomalenvironment or in the caveolear environment). In some aspects, thetherapeutic agent can also be attached to the antibody with anon-cleavable linker

Typically the ADC comprises a linker region between the therapeuticagent and the anti-NTB-A antibody. As noted supra, typically, the linkercan be cleavable under intracellular conditions, such that cleavage ofthe linker releases the therapeutic agent from the antibody in theintracellular environment (e.g., within a lysosome or endosome orcaveolea). The linker can be, e.g., a peptidyl linker that is cleaved byan intracellular peptidase or protease enzyme, including a lysosomal orendosomal protease. Cleaving agents can include cathepsins B and D andplasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123,1999). Most typical are peptidyl linkers that are cleavable by enzymesthat are present in NTB-A-expressing cells. For example, a peptidyllinker that is cleavable by the thiol-dependent protease cathepsin-B,which is highly expressed in cancerous tissue, can be used (e.g., alinker comprising a Phe-Leu or a Val-Cit peptide).

The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysisat certain pH values. Typically, the pH-sensitive linker is hydrolyzableunder acidic conditions. For example, an acid-labile linker that ishydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone,thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or thelike) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805;5,622,929; Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999;Neville et al., Biol. Chem. 264:14653-14661, 1989.) Such linkers arerelatively stable under neutral pH conditions, such as those in theblood, but are unstable at below pH 5.5 or 5.0, the approximate pH ofthe lysosome.

Other linkers are cleavable under reducing conditions (e.g., a disulfidelinker) Disulfide linkers include those that can be formed using SATA(N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., Cancer Res. 47:5924-5931,1987; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987. See also U.S. Pat. No. 4,880,935.)

The linker can also be a malonate linker (Johnson et al., AnticancerRes. 15:1387-93, 1995), a maleimidobenzoyl linker (Lau et al.,Bioorg-Med-Chem. 3:1299-1304, 1995), or a 3′-N-amide analog (Lau et al.,Bioorg-Med-Chem. 3:1305-12, 1995).

The linker also can be a non-cleavable linker, such as anmaleimido-alkylene- or maleimide-aryl linker that is directly attachedto the therapeutic agent and released by proteolytic degradation of theantibody.

Typically, the linker is not substantially sensitive to theextracellular environment, meaning that no more than about 20%,typically no more than about 15%, more typically no more than about 10%,and even more typically no more than about 5%, no more than about 3%, orno more than about 1% of the linkers in a sample of the ADC is cleavedwhen the ADC is present in an extracellular environment (e.g., inplasma). Whether a linker is not substantially sensitive to theextracellular environment can be determined, for example, by incubatingindependently with plasma both (a) the ADC (the “ADC sample”) and (b) anequal molar amount of unconjugated antibody or therapeutic agent (the“control sample”) for a predetermined time period (e.g., 2, 4, 8, 16, or24 hours) and then comparing the amount of unconjugated antibody ortherapeutic agent present in the ADC sample with that present in controlsample, as measured, for example, by high performance liquidchromatography.

The linker can also promote cellular internalization. The linker canpromote cellular internalization when conjugated to the therapeuticagent (i.e., in the milieu of the linker-therapeutic agent moiety of theADC or ADC derivate as described herein). Alternatively, the linker canpromote cellular internalization when conjugated to both the therapeuticagent and the anti-NTB-A antibody (i.e., in the milieu of the ADC asdescribed herein).

Exemplary antibody-drug conjugates include auristatin basedantibody-drug conjugates meaning that the drug component is anauristatin drug. Auristatins bind tubulin, have been shown to interferewith microtubule dynamics and nuclear and cellular division, and haveanticancer activity. Typically the auristatin based antibody-drugconjugate comprises a linker between the auristatin drug and theanti-NTB-A antibody. The linker can be, for example, a cleavable linker(e.g., a peptidyl linker) or a non-cleavable linker (e.g., linkerreleased by degradation of the antibody). The auristatin can beauristatin E or a derivative thereof. The auristatin can be, forexample, an ester formed between auristatin E and a keto acid. Forexample, auristatin E can be reacted with paraacetyl benzoic acid orbenzoylvaleric acid to produce AEB and AEVB, respectively. Other typicalauristatins include MMAF, and MMAE. The synthesis and structure ofexemplary auristatins are described in U.S. Pat. Nos. 7,659,241,7,498,298, 2009-0111756, 2009-0018086, and 7,968,687 each of which isincorporated herein by reference in its entirety and for all purposes.

Exemplary auristatin based antibody drug conjugates include vcMMAE,vcMMAF and mcMMAF antibody drug conjugates as shown below wherein Ab isan antibody as described herein and val-cit represents thevaline-citrulline dipeptide:

or a pharmaceutically acceptable salt thereof. The drug loading isrepresented by p, the number of drug-linker molecules per antibody.Depending on the context, p can represent the average number ofdrug-linker molecules per antibody, also referred to the average drugloading. P ranges from 1 to 20 and is preferably from 1 to 8. In somepreferred embodiments, when p represents the average drug loading, pranges from about 2 to about 5. In some embodiments, p is about 2, about3, about 4, or about 5. The average number of drugs per antibody in apreparation may be characterized by conventional means such as massspectroscopy, HIC, ELISA assay, and HPLC. In some aspects, the anti-NTBAantibody is attached to the drug-linker through a cysteine residue ofthe antibody. In some aspects, the cysteine residue is one that isengineered into the antibody. In other aspects, the cysteine residue isan interchain disulfide cysteine residue.

VI. Applications

In another aspect, the present invention provides methods of using ananti-NTBA-antibody as described herein to modulate a biological activityof an NTB-A-expressing cell, including, for example, natural killer (NK)cells, NK-like T-cells, T-cells, monocytes, dendritic cells, B-cells,and eosinophils. Such methods include, for example, methods ofinhibiting an activity of an NTB-A-expressing cell (e.g., inhibitingcell proliferation). Such methods further include, e.g., methods fortreatment of a disease or disorder associated with an NTB-A-expressingcell.

For example, the anti-NTBA antibodies of the present invention, as nakedantibodies or as antibody drug conjugates thereof, can be used to treatan NTB-A-expressing cancer. Some such cancers show detectable levels ofNTB-A measured at either the protein (e.g., by immunoassay using one ofthe exemplified antibodies) or mRNA level. Some such cancers showelevated levels of NTB-A relative to noncancerous tissue of the sametype, preferably from the same patient. An exemplary level of NTB-A oncancer cells amenable to treatment is 5000-150000 NTB-A molecules percell, although higher or lower levels can be treated. Optionally, alevel of NTB-A in a cancer is measured before performing treatment.

Examples of cancers associated with NTB-A expression and amenable totreatment include hematological malignancies, including B-cell, T-cell,and NK-cell malignancies. In some embodiments, the cancer is a multiplemyeloma, an acute myeloid leukemia (AML), a chronic lymphocytic leukemia(CLL), or a T-Cell or B-cell lymphoma such as, e.g., a non-Hodgkin'slymphoma (NHL). The treatment can be applied to patients having primaryor metastatic tumors of these kinds. The treatment can also be appliedto patients who are refractory to conventional treatments, or who haverelapsed following a response to such treatments.

In a related aspect, the present invention provides a method of treatingmultiple myeloma using an antibody-drug conjugate (ADC) comprising anantibody that specifically binds to NTB-A. In certain variations of thisaspect, an anti-NTB-A ADC for treating multiple myeloma comprises ananti-NTB-A antibody as described herein (e.g., a humanized antibodycomprising VH and VL domains respectively derived from a VH domainhaving the amino acid sequence as shown in residues 20-135 of SEQ IDNO:1 and a VL domain having the amino acid sequence as shown in residues21-140 of SEQ ID NO:2). In other aspects, an anti-NTB-A ADC for treatingmultiple myeloma comprises an antibody other than an antibody asdescribed herein that specifically binds to an extracellular domain ofNTB-A. A collection of anti-NTB-A antibodies are known in the art.Additional antibodies to anti-NTB-A can be made de novo, for example, byimmunizing with NTB-A or one or more extracellular domains thereof. Suchan immunogen can be obtained from a natural source, by peptide synthesisor by recombinant expression. Human antibodies against NTB-A can beprovided by a variety of techniques known in the art.

The anti-NTBA antibodies of the present invention, as naked antibodiesor as antibody drug conjugates thereof, can be used to treat diseasesand disorders associated with B cells, e.g., those diseasescharacterized by excessive numbers of B cells, overactive B cells, ordysfunctional B cells. These diseases include inflammatory diseases andautoimmune disease. Exemplary diseases treatable by the present methodsinclude rheumatoid arthritis, systemic lupus erythematosus, multiplesclerosis, inflammatory bowel disease, asthma, allergy, celiac disease,graft-versus-host disease, and transplant rejection.

The present invention encompasses methods of treating the disease anddisorders described herein as a monotherapy or in combination therapywith, for example, standard of care for treatment of such diseasesand/or disorders. Accordingly, methods for the treatment of cancerinclude administering to a patient in need thereof an effective amountof an antibody or antibody drug conjugate of the present invention incombination with an additional anti-cancer agent or other agent toalleviate symptoms of the cancer. Methods for the treatment ofautoimmune disease include administering to a patient in need thereof aneffective amount of an antibody or antibody drug conjugate of thepresent invention in combination with an additional therapeutic agentfor the treatment of autoimmune disease. Methods for the treatment ofinflammatory disease include administering to a patient in need thereofan effective amount of an antibody or antibody drug conjugate of thepresent invention in combination with an additional therapeutic agentfor the treatment of inflammatory disease.

Anti-NTB-A antibodies, alone or as conjugates thereof, are administeredin an effective regime meaning a dosage, route of administration andfrequency of administration that delays the onset, reduces the severity,inhibits further deterioration, and/or ameliorates at least one sign orsymptom of cancer. In some instances, therapeutic efficacy can beobserved in an individual patient relative to historical controls orpast experience in the same patient. In other instances, therapeuticefficacy can be demonstrated in a preclinical or clinical trial in apopulation of treated patients relative to a control population ofuntreated patients.

Exemplary dosages for an anti-NTB-A antibody are 0.1 mg/kg to 50 mg/kgof the patient's body weight, more typically 1 mg/kg to 30 mg/kg, 1mg/kg to 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to 12 mg/kg, or 1 mg/kgto 10 mg/kg′, or 2 mg/kg to 30 mg/kg, 2 mg/kg to 20 mg/kg, 2 mg/kg to 15mg/kg, 2 mg/kg to 12 mg/kg, or 2 mg/kg to 10 mg/kg, or 3 mg/kg to 30mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 12 mg/kg, or3 mg/kg to 10 mg/kg. Exemplary dosages for a monoclonal antibody orantibody drug conjugates thereof are 0.1 mg/kg to 7.5 mg/kg, 0.2 mg/kgto 7.5 mg/kg, 0.5 mg/kg to 7.5 mg/kg, 1 mg/kg to 7.5 mg/kg, or 2 mg/kgto 7.5 mg/kg or 3 mg/kg to 7.5 mg/kg of the subject's body weight, or0.1-20, or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 mg/kg) or 10-1500 or 200-1500 mg as a fixed dosage. In somemethods, the patient is administered a dose of at least 0.3 mg/kg, 0.5mg/kg, 1 mg/kg 1.5 mg/kg, at least 2 mg/kg or at least 3 mg/kg,administered once every three weeks or greater. The dosage depends onthe frequency of administration, condition of the patient and responseto prior treatment, if any, whether the treatment is prophylactic ortherapeutic and whether the disorder is acute or chronic, among otherfactors.

Administration is typically parenteral. Administration can also belocalized directly into a tumor. Administration into the systemiccirculation by intravenous or subcutaneous administration is preferred.Intravenous administration can be, for example, by infusion over aperiod such as 30-90 min or by a single bolus injection.

The frequency of administration depends on the half-life of the antibodyor conjugate in the circulation, the condition of the patient and theroute of administration among other factors. The frequency can be daily,weekly, monthly, quarterly, or at irregular intervals in response tochanges in the patient's condition or progression of the cancer beingtreated. An exemplary frequency for intravenous administration isbetween twice a week and quarterly over a continuous course oftreatment, although more or less frequent dosing is also possible. Otherexemplary frequencies for intravenous administration are between weeklyor three out of every four weeks over a continuous course of treatment,although more or less frequent dosing is also possible. For subcutaneousadministration, an exemplary dosing frequency is daily to monthly,although more or less frequent dosing is also possible.

The number of dosages administered depends on the nature of the cancer(e.g., whether presenting acute or chronic symptoms) and the response ofthe disorder to the treatment. For acute disorders or acuteexacerbations of a chronic disorder between 1 and 10 doses are oftensufficient. Sometimes a single bolus dose, optionally in divided form,is sufficient for an acute disorder or acute exacerbation of a chronicdisorder. Treatment can be repeated for recurrence of an acute disorderor acute exacerbation. For chronic disorders, an antibody can beadministered at regular intervals, e.g., weekly, fortnightly, monthly,quarterly, every six months for at least 1, 5 or 10 years, or the lifeof the patient.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. The concentration of antibody in a liquid formulation can bee.g., 1-100 mg/ml, such as 10 mg/ml.

Treatment with antibodies of the invention can be combined withchemotherapy, radiation, stem cell treatment, surgery other treatmentseffective against the disorder being treated. Useful classes of otheragents that can be administered with an anti-NTB-A antibody include, forexample, antibodies to other receptors expressed on cancerous cells,antitubulin agents (e.g., auristatins), DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, and the like.

Treatment with the anti-NTB-A antibody, optionally in combination withany of the other agents or regimes described above alone or as anantibody drug conjugate, can increase the median progression-freesurvival or overall survival time of patients with an NTB-A-expressingcancer (e.g., multiple myeloma, AML, NHL), especially when relapsed orrefractory, by at least 30% or 40% but preferably 50%, 60% to 70% oreven 100% or longer, compared to the same treatment (e.g., chemotherapy)but without an anti-NTB-A antibody alone or as a conjugate. In additionor alternatively, treatment (e.g., standard chemotherapy) including theanti-NTB-A antibody alone or as a conjugate can increase the completeresponse rate, partial response rate, or objective response rate(complete+partial) of patients with an NTB-A-expressing cancer by atleast 30% or 40% but preferably 50%, 60% to 70% or even 100% compared tothe same treatment (e.g., chemotherapy) but without the anti-NTB-Aantibody.

Typically, in a clinical trial (e.g., a phase II, phase II/III or phaseIII trial), the aforementioned increases in median progression-freesurvival and/or response rate of the patients treated with standardtherapy plus the anti-NTB-A antibody, relative to the control group ofpatients receiving standard therapy alone (or plus placebo), arestatistically significant, for example at the p=0.05 or 0.01 or even0.001 level. The complete and partial response rates are determined byobjective criteria commonly used in clinical trials for cancer, e.g., aslisted or accepted by the National Cancer Institute and/or Food and DrugAdministration.

In other applications, the anti-NTB-A antibodies of the presentinvention can be used for detecting NTB-A in the context of clinicaldiagnosis or treatment or in research. Expression of NTBA on a cancerprovides an indication that the cancer is amenable to treatment with theantibodies of the present invention. The antibodies can also be sold asresearch reagents for laboratory research in detecting cells bearingNTB-A and their response to various stimuli. In such uses, an anti-NTB-Aantibody can be labeled with a fluorescent molecule, a spin-labeledmolecule, an enzyme, or a radioisotype, and can be provided in the formof kit with all the necessary reagents to perform the assay for NTB-A.The antibodies can also be used to purify NTB-A, e.g., by affinitychromatography.

All patent filings, other publications, accession numbers and the likecited above or below are incorporated by reference in their entirety forall purposes to the same extent as if each individual item werespecifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publicationare published at different times, the version most recently published atthe effective filing date of the application is meant unless otherwiseindicated. Any feature, step, element, embodiment, or aspect of theinvention can be used in combination with any other unless specificallyindicated otherwise. Although the present invention has been describedin some detail by way of illustration and example for purposes ofclarity and understanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.

Examples Example 1: Expression of NTB-A on Multiple Myeloma Cell Lines

Amo-1, JJN-3, Karpas-620, KMS-12-BM, MOLP-8, OPM-2 (DSMZ; RPMI 1640+20%PBS), L-363 (DSMZ; RPMI 1640+15% FBS), LP-1, SK-MM-2 (DSMZ; RPMI1640+10% FBS), EJM (DSMZ; EMEM+20% FBS), MM.1R, MM.1S, NCI-H929,RPMI-8226 (ATCC; RPMI 1640+10% FBS), and U-266 (ATCC; RPMI 1640+15% FBS)cell lines were cultured at 37° C., 5% CO2. 500,000 cells were stainedin FACS buffer (PBS+3% FBS+0.02% sodium azide) for 45 minutes on icewith anti-NTB-A antibodies NT-7 and 11A1. A PE-conjugated secondaryantibody was used for detection. Stained cells were analyzed using aFACSCalibur flow cytometer (Becton Dickinson).

NTB-A was shown to be expressed on five of fifteen multiple myeloma celllines, Karpas620, EJM, MM.1R, MM.1S and U-266.

Example 2: Expression of NTB-A on Multiple Myeloma Patient Samples

Frozen human multiple myeloma patient bone marrow aspirate samples (5-10million cells) were purchased from BioServe (Beltsville, Md.), AllCells(Emeryville, Calif.), Conversant Healthcare Systems (Huntsville, Ala.),and ProteoGenex (Culver City, Calif.). MM patient bone marrow cells werethawed at 37° C., transferred into prewarmed RPMI 1640 media, andtreated with DNase 1 (0.05 mg/mL) for 10 minutes at room temperature tominimize cell aggregation. The cells were then centrifuged (1,400 rpm; 5minutes), resuspended into RPMI 1640+10% FBS), and cell number/viabilitymeasured by trypan blue exclussion. Next, the patient cells werecentrifuged, resuspended into FACS buffer (PBS+3% FBS, +0.02% sodiumazide) on ice, filtered through a 100 μm cell strainer to remove anydebris, and 100 μL cell suspension aliquoted into the wells of aV-bottom 96-well plate for staining (1.2×105-1.0×106 viable cells/well).The patient bone marrow aspirate cells were triple stained withanti-hCD38-FITC, anti-hCD45-APC and PE-conjugated antiNTB-A (clone:NT-7) or isotype control IgG (30 minutes on ice). Stained cells werewashed twice in FACS buffer and analyzed by flow cytometry using theFACsCalibur flow cytometer. Cell surface expression of NTB-A wasquantitated for the multiple myeloma cell CD38+/CD45-gatedsubpopulation. Expression of CD138 (clone:ID4) positive control multiplemyeloma antigen was also measured.

NTB-A was shown to be expressed on the surface of 13 out of 15 patientsamples. See Table 3 below:

TABLE 3 Results Summary for NTB-A Expression on Human Multiple MyelomaPatient Plasma Cells Multiple NTB-A CD138 Myeloma Expression ExpressionPatient Treatment History (clone: NT-7) (clone: 1D4) 1 Stage III;relapsed 2.37 1.51 2 Newly diagnosed 15.4 7.99 3 Newly diagnosed 79.8143 4 VAD chemotherapy; 25.0 745 Autologous transplant 5 VADchemotherapy 14.0 12.8 6 Bone marrow transplant; 1.16 1.65 Bortezomib 7Newly diagnosed 18.2 1.45 8 Newly diagnosed 87.3 46.6 9 Newly diagnosed85.7 17.3 10 Chemotherapy; 1.22 106 Autologous transplant 11 Melphalan,Lenalidomide 5.09 2.20 12 Lenalidomide, Cytoxan, 7.56 3.25 Thalidomide13 Bortezomib 17.3 4.55 14 VAD chemotherapy; 17.8 74.33 Lenalidomide,Bortezomib 15 Newly Diagnosed 12.7 11.45 Fold increase of antigenexpression mean fluorescence intensity) over isotype control antibody

Example 3: Antibody Selection

Lymphocytes harvested from spleen and lymph nodes of NTB-A antibodyproducing mice were fused to myeloma cells. Fused cells were recoveredovernight in hybridoma growth media. Following recovery, cells were spundown and then plated in semi-solid media. Hybridomas were incubated andIgG producing hybridoma clones were picked. Hybridoma culturesupernatants were screened and 313 out of 478 hybridoma clones werefound to specifically bind to NTB-A extracellular domain by measuringfluorescent signal on the surface of NTB-A positive cells. Specificbinding of fluorescent labelled ADCs to extracellular domain NTB-A wasconfirmed by flow cytometry (BD Biosciences FACSCalibur) using a panelof NTB-A expressing multiple myeloma cells at 2.0 ocg/ml concentration.The 313 hybridomas that bound to NTB-A were expanded for directconjugation with vc-MMAE using the methods described in InternationalApplication No. WO 2011/109308. The directly conjugated antibody panelwas tested in binding and cytotoxicity assays. The 313 NTBA-A bindingADCs were screened for cytotoxicity with multiple myeloma cell lines.Cytoxicity studies were done by plating 15,000 multiple myeloma cellsper well in the appropriate growth media. For cell-based binding assays,anti-NTB-A vcMMAE 4-loaded antibody drug conjugates were tested at 12.5,50.0, and 200 ng/mL final concentration on cells and incubated for 96hours total at 37° C. Cell viability was measured using the Cell TiterGlo (Promega) luminescence assay and the potency of ADCs was assessedbased on the percent viability relative to untreated control cells. IC₅₀values were generated from dose curves produced using Prism software(GraphPad). The cytotoxicity cut-off was set at an IC₅₀ less than orequal to 50 ng/ml. The 69 most potent anti-NTBA monoclonal antibodies asADCs were moved forward. Only 6 of the 69 most potent ADCs demonstratedan IC₅₀ value less than 12.5 ng/ml. Saturation binding curves and Kdvalues were determined by flow cytometry for a small panel of the highlycytotoxic anti-NTB-A ADCs. Anti-NTB-A ADCs with the 11A1 and 26B7antibodies were determined to be the most cytotoxic.

Example 4: Anti-NTB-A Internalization Assay

The murine 11A1 antibody mcMMAF conjugate was evaluated for its abilityto internalize in the NTB-A⁺ cell line U-266 (FIG. 1).

U-266 antigen-positive cells were incubated with 5 μg/mL anti-humanNTB-A antibody drug conjugate, 11A1-mcMMAF (the antimitotic agent MMAFconjugated via a maleimidocaproyl linker (mc) to a stoichiometry of 4drugs per antibody via cysteine linkages) for 30 minutes on a shaker at4° C. Cells were washed three times with RPMI 1640 media+10% fetalbovine serum and then plated out at 5×105 cells/100 μL, per well intotwo identical 96-well U-bottom plates (BioSciences, San Jose, Calif.).One plate was placed at 37° C. and the other at 4° C. One sample wasimmediately washed and stained for time zero. Cells were collected fromboth plates at 0.5, 1, 2, 8, and 24 hour time points, washed two timeswith cold wash buffer (PBS+2.5% fetal bovine serum), and stained with 10μg/mL goat-anti-mouse IgG-PE (Jackson ImmunoResearch, West Grove, Pa.)for 30 minutes, on ice and protected from light. Cells were again washedtwice with wash buffer and fixed with 500 μL of PBS+1% paraformaldehyde.Once all samples were collected and stained, they were analyzed on theFACs Calibur flow cytometer (BD BioSciences, San Jose, Calif.), and datawas expressed as a percentage of time zero MFI.

Example 5: ADC Cytotoxicity Assays

Antibody-drug conjugates (ADCs) were prepared for the murine anti-NTB-Amonoclonal antibodies 11A1 and 26B7. The antimitotic agent monomethylauristatin E (MMAE) was conjugated to anti-NTB-A mAbs via acathepsin-cleavable valine-citrulline (vc) linker and monomethylauristatin F (MMAF) was conjugated via a maleimidocaproyl (mc) linker toa stoichiometry of 4 drugs per antibody via cysteine linkage asdescribed in U.S. Pat. Nos. 7,659,241 and 7,498,298.Anti-NTB-Avc-MMAE(4) and -mc-MMAF(4) ADCs were serially diluted 3-foldin media to produce a 10 point dose curve (1,000 ng/mL-0.05081 ng/mL)and applied to multiple myeloma cells cultured in 96-well assay plates.Karpas-620, EJM, MM.1R, MM.1S, U-266 (NTB-A+), and L363 (NTB-A−)multiple myeloma cell lines were treated with anti-NTB-A ADCs inquadruplicate and incubated for 96 hours at 37° C., 5% CO2. Cells wereassayed for viability using the Cell Titer Glo luminescent cytotoxicityassay (Promega), and data collected using an EnVision plate reader(Perkin Elmer). Dose effect curves and IC50 values were calculated usingGraphPad Prism software.

TABLE 4 Results Summary for ADC Cytotoxicity Assay against multiplemyeloma cell lines Karpas- U- Ab Drug 620 EJM MM. 1S 266 MM.1R Isotype11A1 vcMMAE 3.77 12.5 48.9 2.37 13.6 mIgG1 11A1 mcMMAF 0.648 2.48 3.100.877 2.52 mIgG1 26B7 vcMMAE 431 52.5 >1000 6.04 78.9 mIgG1 26B7 mcMMAF2.61 6.07 3.99 1.72 5.86 mIgG1

TABLE 5 Results Summary for ADC Cytotoxicity Assay against AML and NHLcell lines Ramos HNT-34 HEL92.1.7 KG-1 Ab Drug CA46 (NHL) (NHL) (AML)(AML) (AML) Isotype 11A1 vcMMAE 8.91 5.77 1.58 >1000 14.0 mIgG1 11A1mcMMAF 2.61 1.76 0.861 >1000 2.09 mIgG1 26B7 vcMMAE 13.8 6.83 3.26 >1000178.2 mIgG1 26B7 mcMMAF 4.91 3.19 1.13 >1000 2.45 mIgG1

Example 6: Competitive Binding Assay

The assay described in this example details the method used to evaluatethe ability of a sample antibody to compete for binding with the 11A1murine antibody. For this particular study, the ability of the 26B7antibody and the NT-7 antibody (clone: NT-7 (Biolegend #317208) tocompete with the 11A1 antibody was evaluated. Also evaluated was theability of the 11A1 antibody to compete with itself. The assay utilizesa “reference antibody” comprising the murine 11A1 IgG1 antibody (i.e.,VH and VL domains in the bivalent structure of a native (natural)antibody, i.e., a tetramer consisting of two identical pairs ofimmunoglobulin chains, each pair having one light chain and one heavychain.)

Exponentially growing NTB-A positive cells expressing about 24,500 NTB-Asurface molecules per cell (e.g., Ramos cells) was collected and washedin isotonic phosphate buffered saline (PBS) and stored on ice. The NTB-Apositive cells were aliquoted into wells of a 96-well v-bottom plate onice, 2×10⁵ cells per well in 100 μL volume per well.

A 2× concentration of the reference antibody conjugated to a fluorescentlabel (e.g., AF647) was prepared in PBS/FBS (PBS containing 2% fetalbovine serum (FBS)/0.02% sodium azide), with the 1× concentration beingequal to 0.0188 μg/mL (the Kd value concentration of 11A1 mAb). ThisPBS/FBS/Labeled-Ab solution was then aliquoted into wells of a 96-welldilution plate (200 μL/well). Row A of wells was left for the initial 2×mix of unlabeled sample antibody in the PBS/FBS/Labeled-Ab solution (seeitem (4) below); sufficient wells were also left for controls.

A 2× concentration of an unlabeled sample antibody (to be evaluated forcompetitive binding) was prepared in the PBS/FBS AF Labeled-Ab solution(20 μg/mL of sample antibody; therefore, the 1× concentration equal to10 μg/mL). The 2× concentration of unlabeled sample antibody was thenaliquoted to wells in the first column of the 96-well dilution plate.Samples were then serially diluted in a five-fold dilution series (50μL, from the initial sample dilution is added to the 200 μL of thePBS/FBS/Labeled-Ab solution in the next row, repeating from row to rowof the plate).

Sufficient wells (e.g., wells A1 through A6) were left for flowcytometry set up and unstained wells, with PBS/FBS only added (nolabeled reference antibody). These wells were used as the unstainedcontrols (0% staining). Sufficient wells (e.g., wells A7 though A12)were also left to serve as the 100% staining and receive thePBS/FBS/Labeled-Ab solution without unlabeled sample antibody.

100 μL of diluted samples from the dilution plate were then transferredin duplicate to corresponding wells of cells (100 μL) in the v-bottomplate to yield 1× concentrations. These samples were then incubated forone hour on ice protected from light.

Cells were then washed twice with PBS/FBS. For example, the plate wasspun down at 1000 rpm for 5 minutes and the supernatant discarded,plates vortexed to disperse cells, and wash buffer (PBS/FBS) added(about 200 μL/well/wash); after the last spin, the plate was invertedand blotted gently.

Following the wash, cell pellets were resuspended in 250 μL of PBS/FBSand the cells were kept at 2-8° C., protected from light, until analyzedon a flow cytometer (e.g., an LSRII flow cytometer, BD BioSciences, SanJose, Calif.).

Once on the flow cytometer, the cell population of interest was isolatedby gating on forward scatter and side scatter populations (FSC/SSC) toyield population plots, and the fluorescence signal for the fluorescentlabel was acquired.

The flow cytometry data was then analyzed using a sigmoidaldose-response analysis (variable slope). The IC50 of the unlabeledantibody was determined from the fitted curve (i.e., the concentrationof the unlabeled sample antibody at which the labeled reference antibodyexhibits 50% of maximum binding).

The 11A1 antibody competed with itself and with antibody 26B7 but notwith antibody NT-7. Only those antibodies that reduce 11A1 binding toless than 45% of maximum binding at a concentration of 10 ug/ml ofunlabeled sample antibody (preferably less than 20% or even less than10%) are deemed to compete with 11A1 for binding to NTB-A.

The 11A1 and 26B7 antibodies were also tested for competition with the480.12 antibody using FACS-based competition assays as described in U.S.Pat. No. 7,874,067. The 11A1 and 26B7 antibodies did not compete forbinding with the 480.12 antibody in such assays (data not shown).

Example 7: mAb Affinity Measurements and Binding Specificity

Dose titrations of the murine anti-human NTB-A antibodies conjugated toAlexa Fluor 647 (11A1 and 26B7) were used to generate a saturationbinding curve. Antigen positive Ramos cells were plated at 1×10⁵ cellsper well in a 96 well V-bottom plate (Thermo Scientific, Rochester,N.Y.). Three-fold serial dilutions of 2× concentrated antibodies wereprepared in FACs buffer (PBS+2% fetal bovine serum+0.02% azide) and wereadded to the cells in duplicate. The antibody solutions were incubatedwith cells for 1 hour on ice, protected from light. The cells werewashed twice with FACs buffer and analyzed on the LSRII flow cytometer(BD BioSciences, San Jose, Calif.). KD values were determined withGraphPad Prism software (La Jolla, Calif.).

Murine anti-human NTB-A antibodies 11A1 and 26B7 were tested for bindingto U-266 multiple myeloma cells and stable transfectants of293-F17/hNTB-A isoform 4 and 293-F17/cynomolgus-NTB-A. Cells were platedat 2.5×105 cells/well in FACS buffer (PBS+2% fetal bovine serum+0.02%azide) and incubated with 2 μg/mL antibody for 45 minutes on ice. Thecells were washed twice and stained with goat anti-mouse IgG-PE (JacksonImmunoResearch, West Grove, Pa.) for 30 minutes, on ice and protectedfrom light. Cells were again washed twice with FACs buffer and fixedwith 500 μL of 1×PBS+1% paraformaldehyde. Stained cells were analyzed onthe FACs Calibur flow cytometer (BD BioSciences, San Jose, Calif.).

TABLE 6 Affinity Measurements Kd (nM) Antibody Cell line Human NTB-A11A1 murine IgG1 antibody Ramos 0.13 26B7 murine IgG1 antibody Ramos0.16

Example 8: In Vivo MM Xenograft Studies

NOD scid IL2 receptor gamma chain knockout (NSG) mice are used todevelop disseminated cell line models of multiple myeloma in which tumorcells localize to the bone marrow compartment. Mice are implanted with 1million MM.1R multiple myeloma cells or 5 million U-266 multiple myelomacells, then dosed 5 days after tumor cell implant with anti-NTB-Aantibody drug conjugates. The vcMMAE anti-NTB-A antibody drug conjugatesloaded with 4 vcMMAE molecules are delivered to mice throughintraperitoneal injection in a single dose at 1.0 and 3.0 mg/kg. Mouseserum is monitored by ELISA assay for levels of circulating lambda lightchain Ig, secreted by the multiple myeloma tumor cells, every 2 to 3weeks post antibody drug conjugate dosing. Mice in each study group areevaluated for signs of disease progression, and morbidity, andsacrificed on advanced signs of disease. Kaplan-Meier survival plots aregenerated for control and treatment study groups, and statisticalanalysis run to determine the significance of the observed antitumoractivity at each ADC dose level versus vehicle control or nonbindingcontrol ADC.

What is claimed is:
 1. A method of treating a patient having a cancercharacterized by NTB-A (SLAMF6) expression, the method comprising:administering to the patient an effective regime of a monoclonalantibody that specifically binds to human NTB-A, wherein the antibodycomprises a CDR-H1 amino acid sequence as shown in SEQ ID NO: 5 or SEQID NO: 11, a CDR-H2 amino acid sequence as shown in SEQ ID NO: 6 or SEQID NO: 12, a CDR-H3 amino acid sequence as shown in SEQ ID NO: 7 or SEQID NO: 13, a CDR-L1 amino acid sequence as shown in SEQ ID NO: 8 or SEQID NO: 14, a CDR-L2 amino acid sequence as shown in SEQ ID NO: 9 or SEQID NO: 15, and a CDR-L3 amino acid sequence as shown in SEQ ID NO: 10 orSEQ ID NO:
 16. 2. The method of claim 1, wherein the cancer is selectedfrom the group consisting of multiple myeloma, acute myeloid leukemia(AML), and a T or B-cell lymphoma.
 3. The method of claim 2, wherein theB-cell lymphoma is non-Hodgkin's lymphoma (NHL).